Bicuculline, a GABAA-receptor antagonist, blocked HPA axis activation induced by ghrelin under an acute stress.

Ghrelin is a peptide of 28 amino acids with a homology between species, which acts on the central nervous system to regulate different actions, including the control of growth hormone secretion and metabolic regulation. It has been suggested that central ghrelin is a mediator of behavior linked to stress responses and induces anxiety in rodents and birds. Previously, we observed that the anxiogenic-like behavior induced by ghrelin injected into the intermediate medial mesopallium (IMM) of the forebrain was blocked by bicuculline (a GABAA receptor competitive antagonist) but not by diazepam (a GABAA receptor allosteric agonist) in neonatal meat-type chicks (Cobb). Numerous studies have indicated that hypothalamic-pituitary-adrenal (HPA) axis activation mediates the response to stress in mammals and birds. However, it is still unclear whether this effect of ghrelin is associated with HPA activation. Therefore, we investigated whether anxiety behavior induced by intra-IMM ghrelin and mediated through GABAA receptors could be associated with HPA axis activation in the neonatal chick. In the present study, in an Open Field test, intraperitoneal bicuculline methiodide blocked anxiogenic-like behavior as well as the increase in plasma ACTH and corticosterone levels induced by ghrelin (30pmol) in neonatal chicks. Moreover, we showed for the first time that a competitive antagonist of GABAA receptor suppressed the HPA axis activation induced by an anxiogenic dose of ghrelin. These results show that the anxiogenic ghrelin action involves the activation of the HPA axis, with a complex functional interaction with the GABAA receptor.

Gabaergic control of anxiety-like behavior, but not food intake, induced by ghrelin in the intermediate medial mesopallium of the neonatal chick.

Ghrelin (Grh) is an endogenous ligand of the growth hormone secretagogue receptor. In neonatal chicks, central Ghr induces anxiogenic-like behavior but strongly inhibits food intake. The intermediate medial mesopallium (IMM) of the chick forebrain has been identified to be a site of the memory formation, and the modulation of the GABAA receptors that are present here modifies the expression of behavior. Thus, the GABAergic system may constitute a central pathway for Ghr action in regulating the processes of food intake and stress-related behaviors. Therefore, we investigated if the effect of systemic administration of bicuculline (GABAA receptor antagonist) and diazepam (benzodiazepine receptor agonist) on the anxiety in an Open Field test and inhibition in food intake induced by Grh (30pmol) when injected into IMM, were mediated by GABAergic transmission. In Open Field test, bicuculline was able to block the anxiogenic-like behavior induced by Ghr, whereas diazepam did not produce it. However, the co-administration of bicuculline or diazepam plus Ghr did not show any change in food intake at 30, 60 and 120min after injection compared to Ghr alone. Our results indicate for the first time that Ghr, injected into the forebrain IMM area, induces an anxiogenic-like behavior, which was blocked by bicuculline but not diazepam, thus suggesting that Ghr plays an important role in the response pattern to acute stressor, involving the possible participation of the GABAergic system. Nevertheless, as neither drug affected the hypophagia induced by intra-IMM Ghr, this suggests that it may be mediated by different mechanisms.

Central α- and ß-thujone: similar anxiogenic-like effects and differential modulation on GABAA receptors in neonatal chicks.

The convulsant effects of α-thujone are attributed to inhibitory actions on the GABAA receptor. We investigated, for the first time, the effects of α-thujone or ß-thujone administrated centrally on the fear/anxiety behaviour of 3-day-old chicks in an Open Field and their modulation on the GABAA receptor. Higher doses were convulsant by eliciting a toxic and excitatory action, with the results showing that a dose of 78 nmol of either of the two diastereoisomers had an anxiogenic-like effect observed as an increased latency to ambulate and a reduced locomotor activity in an Open Field. Nevertheless, only the central administration of α-thujone reversed the increase induced by acute stress in the flunitrazepam-sensitive GABAA receptor recruitment. These findings demonstrated that α-thujone, when intracerebroventricularly administered, suppressed the GABAA receptor recruitment induced by acute stress, maybe due to α-thujone blocking the benzodiazepine binding site or another site of the GABAA complex. However, it should not be discarded that acute stress associated with novelty may have induced the recruitment of a subpopulation of GABAA receptors more sensitive to α-thujone than to the constitutive receptors, or that this monoterpene could have inhibited any protein or enzyme trafficking that modulated the phosphorylation of the receptor involved in the turnover of GABAA receptor. ß-Thujone showed behavioural effects similar to its diastereoisomer α-thujone. However, its action mechanism may have been mediated by other neurotransmitter systems, such as the serotonergic one or by a different biological effectiveness due to a distinct stereochemistry at the specific site of the GABAA receptor.

Pharmacological alterations that could underlie radiation-induced changes in associative memory and anxiety.

It is widely known that ionizing radiation is a physical agent broadly used to kill tumor cells during human cancer therapy. Unfortunately, adjacent normal tissues can concurrently undergo undesirable cell injury. Previous data of our laboratory demonstrated that exposure of developing rats to ionizing radiations induced a variety of behavioral differences respect to controls, including changes in associative memory and in anxiety state. However, there is a lack of data concerning modifications in different related pharmacological intermediaries. Therefore, the aim of the present study was to investigate whether the behavioral differences observed in young animals irradiated at birth might be underlain by early changes in PKCß1 levels which, in turn, could lead to changes in hippocampal GABAergic neurotransmission. Male Wistar rats were irradiated with 5Gy of X rays between 24 and 48 h after birth. Different pharmacological markers related to the affected behavioral tasks were assessed in control and irradiated hippocampus at 15 and 30 days, namely GABAA receptor, GAD65-67, ROS and PKCß1. Results showed that all measured parameters were increased in the hippocampus of 30-days-old irradiated animals. In contrast, in the hippocampus of 15-days-old irradiated animals only the levels of PKCß1 were decreased. These data suggest that PKCß1 might constitute a primary target for neonatal radiation damage on the hippocampus. Therefore, it could be hypothesized that an initial decrease in the levels of this protein can trigger a subsequent compensatory increase that, in turn, could be responsible for the plethora of biochemical changes that might underlie the previously observed behavioral alterations.

Participation of the GABAergic system on the glutamate release of frontal cortex synaptosomes from Wistar rats with experimental autoimmune encephalomyelitis.

We previously found that the glutamate release was decreased in synaptosomes from rat cerebral cortex during the development of experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. Various other reports have shown a deficit in the expression of proteins associated with GABAergic neurotransmission in the neocortex of patients with multiple sclerosis and it was also demonstrated that the activation of GABAA receptors leads to an inhibition of glutamate release. Now, in order to evaluate the events that may affect the neuronal function in EAE synaptosomes, we analyzed the participation of the GABAergic system in glutamate release and in the flunitrazepam-sensitive GABAA receptor density. This revealed alterations in the GABAergic system of the frontal cortex synaptosomes from EAE animals. GABA induced a decrease in the 4-aminopyridine-evoked glutamate release in control synaptosomes which was abolished by picrotoxin, a GABAA receptor antagonist. In contrast, synaptosomes from EAE rats showed a loss in the inhibition of glutamate release mediated by GABA. Furthermore, the flunitrazepam-sensitive GABAA receptor density was decreased during the acute stage of the disease in synaptosomes from EAE rats. We also observed a loss of inhibition in the Ca2+-dependent phosphorylation of synapsin I mediated by GABA in nerve terminals from EAE animals, which could explain the loss of GABAergic regulation on evoked glutamate release. The changes observed in the GABAA receptor density as well as the loss of GABAergic inhibition of glutamate release were partially reverted in cortical synaptosomes from recovered EAE animals. These results suggest that the decrease in the flunitrazepam-sensitive GABAA receptor density may explain the observed failure of GABAergic regulation in the glutamate release of synaptosomes from EAE rats, which might contribute to the appearance of clinical symptoms and disease progression.

Effect of neuropeptide-EI on the binding of [3H]SCH 23390 to the dopamine D1 receptor in rat striatal membranes.

We have previously demonstrated that neuropeptide-EI, at high doses, stimulates the production of cAMP, in caudate putamen, through the activation of adenylate cyclase coupled to specific D1 receptors. The aim of the present work was to find evidences for a probable interaction between this neuropeptide and the dopamine D1 receptor in the mammalian central nervous system. The present data show that neuropeptide-EI, at high concentrations, affected both the maximum binding and the apparent affinity of [n-methyl-3H] (R)-(+)-8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate to the dopamine D1 receptor in a concentration-dependent manner.

Day-old chicks categorised on latency to peck, exhibit a stable fear pattern until 15 days of age.

Day-old chicks of both sexes were individually categorised on the latency to peck pebbles and termed as high latency (HL), moderate latency (ML) or low latency (LL). Anxiolytic doses of diazepam diminished the latency only in the HL category, suggesting that it is the most anxious category. At 15 days of age, the LL category showed the lowest latency to ambulate in the open-field test, the lowest immobility duration in the tonic immobility test and insensitivity to anxiolytic doses of diazepam in both behavioural tests suggesting that it is the less anxious category. The increase of the central benzodiazepine receptor density induced by acute stressors was the highest in the most anxious and/or fearful HL category. There were more females than males in the LL category and inversely in the HL category there were more males. The results suggest that the fear pattern depends on the sex and inter-individual differences within a same sex which are stable across life. This could be used as a test for fear and/or anxiety state, and useful to choose fowls with the best performance later in life.

The effect of melanotropic peptides on binding of [(3)H]dihydroalprenolol-hydrochloride to hypothalamic membranes.

In the present work we studied the interaction of alpha-melanocyte-stimulating hormone (alpha-MSH) and ACTH-(1-24) with beta-adrenergic receptors in hypothalamic membranes from rat brain. Saturation curves for [(3)H]dihydroalprenolol-hydrochloride ([(3)H]DHA) binding in the presence of the peptides revealed a decreased binding capacity (Bmax). The dissociation constant (Kd) was, however, not affected by alpha-MSH or ACTH-(1-24). These data indicate a non competitive interaction between these melanocortin peptides and [(3)H]DHA on beta-adrenergic receptors in hypothalamic membranes.

Alpha-melanotropin hormone inhibits the binding of [3H]SCH 23390 to the dopamine D1 receptor in vitro.

We have previously demonstrated that the simultaneous presence of alpha-melanocyte stimulating hormone (alpha-MSH) and dopamine resulted in a reduction in cyclic AMP (cAMP) levels in slices containing caudate putamen and accumbens nuclei as compared to those treated only with dopamine or alpha-MSH. This study was carried out to explore if the interaction between alpha-MSH and dopamine could be explained on the basis of a direct interaction between alpha-MSH and the dopamine D1 receptor. Saturation curves for [n-methyl-3 H](R)-(+)-8 chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 H-3-benzazepin-7-o] hemimaleate ([3H]SCH 23390) binding in the presence of increasing concentrations of alpha-MSH were performed. Nonlinear regression in the presence of alpha-MSH revealed an increased dissociation constant (Kd). The binding capacity (Bmax) was not affected by the peptide. These data suggest an apparent competitive interaction between alpha-MSH and [3H]SCH 23390 in striatal membranes on the dopamine D1 receptor; (Ki = 1.2 X 10(-7) M). The present data show that alpha-MSH could interact with the dopamine D1 receptor modulating allosterically the affinity of [3H]SCH 23390 for the receptor or by causing a change in the lipid environment of the dopamine receptor, resulting in an inhibition of the ligand binding to it.

Learning and novelty induced increase of central benzodiazepine receptors from chick forebrain, in a food discrimination task.

Young chicks were trained to discriminate food grains from inedible pebbles. On Day 1 and Day 2 of the task, latency to peck, and number of pecks were scored and the forebrain [3H]flunitrazepam receptor binding was also determined at 0 and 30 min after an 8-min training session. Compared with quiet controls, the receptor density increased 46%, 30 min after the training session on Day 1. Compared with chicks that had learned the discrimination and were merely repeating already learned behavior on Day 2, the receptor increased more than 46%. Since chicks that had learned the discrimination had a higher behavioral activity, we interpret that the learning of a new task is itself responsible in addition to stress for the receptor density increase. Stressful factors accompanying the learning task as handling and novelty increased 17% the receptor density, 30 min after a training session without food, compared with quiet controls. However, receptor density did not increase in chicks repeating the same housing conditions, suggesting that chicks were habituated to handling and novelty on Day 2. Differences in receptor density were not observed between quiet controls and experimental groups, at 0 min after the training session, indicating that changes were time dependent. In all cases the affinity remained unchanged. Our results suggest that, the GABA(A) receptor (i) is involved in early stages of memory formation and in stress adaptive responses, and (ii) is modulated by new non-repetitive environmental conditions.

Rapid tolerance to benzodiazepine modifies rat hippocampal synaptic plasticity.

Glutamate antagonists to N-methyl-D-aspartate (NMDA) receptors blocks the development of "rapid' tolerance to the sedative action of benzodiazepines (BZDs). This kind of glutamate receptors is closely related to synaptic plasticity in different areas of the brain such as hippocampus. In the present investigation, we studied the synaptic plasticity in dentate gyrus of the hippocampus during the development of tolerance to the hypomotility action induced by diazepam (DZ). The results show an increased hippocampal synaptic plasticity in slices from rats treated with diazepam (5 mg/kg per day) during 4 days, assessed as a decrease of the threshold in the stimulation frequency for long-term potentiation (LTP) elicitation. Thus, a single dose of DZ does not change the ease of induction of LTP but does change locomotor behavior: multiple DZ doses change LTP but not locomotor behavior. Our results reveal a positive correlation between the synaptic plasticity and development of BZD tolerance to locomotor activity.

Chick imprinting performance and susceptibility to acute stress associated to flunitrazepam receptor increase.

One-day-old chicks were selected on their performance in imprinting behaviour and termed high-imprinted (H-I), partially imprinted (P-I) and low-imprinted (L-I) chicks. Then, H-I and L-I chicks were submitted to acute handling stress and [3H]flunitrazepam receptor-binding was performed on synaptosomal membranes from forebrain at various times after handling. The receptor number significantly increased in L-I but not in H-I chicks at 30 min after handling while the affinity remained unchanged at all times. In addition, when the three selected groups were maintained to reach 15 days of age and then they were submitted to acute swimming stress, the degree of receptor increase was also inversely related to the degree of imprinting performance. The receptor increase associated to swimming stress was higher in the left hemisphere, suggesting an interhemispheric asymmetry of stress effects. The results suggest that more-imprinted chicks are less susceptible than less-imprinted chicks to acute stress associated to central benzodiazepine receptor increase, probably due to differences in the degree of endogenous emotionality.

Benzodiazepine receptor recruitment after acute stress in synaptosomal membranes from forebrain of young chicks: action of Triton X-100.

In young chicks submitted to acute stress by forced swimming there was a significant increase in the number of the measurable [3H]-flunitrazepam receptors in synaptosomal membranes from forebrain. In addition, low subsolubilizing concentrations of Triton X-100 caused a significant increase in the measurable [3H]-flunitrazepam receptor number in synaptosomal membranes from non-stressed chicks. However, this Triton X-100 stimulatory effect was not observed when tested in synaptosomal membranes from stressed chicks. In all cases the affinity remained unchanged. This result suggest that: (i) acute stress and Triton X-100 induce receptor recruitment by enhancing [3H]-flunitrazepam accessibility to a pool of receptors which is unmeasurable either before stress or in absence of detergent; (ii) neither recruitment types are additive and they involve receptors coming from the same nonmeasurable pool; (iii) stress induces a maximal recruitment of existing benzodiazepine receptors; (iiii) the pool of nonmeasurable receptors represents about a quarter of the total in control chicks. The recruitment at a short time of stress could be interpreted in terms involving internalization; recycling or modulation of receptors but not its biosynthesis or degradation.