Nancy A. Salvatierra

Central ghrelin increases anxiety in the Open Field test and impairs retention memory in a passive avoidance task in neonatal chicks.

Ghrelin (Grh) is an endogenous ligand for the growth hormone secretagogue receptor. Although Ghr stimulates feeding in rats, it inhibits feeding in neonatal chicks. However, little is known about other central behavioral effects of Ghr. Therefore, we investigated the Ghr effects, injected intracerebroventricularly, on anxiety and memory retention of neonatal chicks in an Open Field test and in a one-trial passive avoidance task, respectively. In the Open Field test, the administration of Ghr in a dose-dependent manner increased the latency to ambulate but decreased ambulation activity, indicating an anxiogenic effect. Furthermore, chicks trained on a passive avoidance task and injected with a dose of 30pmol of Ghr immediately after training showed an impairment of memory retention. However, there were no significant effects on the number of pecks during the pretraining, training, retention and discrimination. In addition, different doses of Ghr produced an inhibition in food intake at different times after injection. Our results indicate that Ghr induces anxiogenesis in chicks. Moreover, we have shown for the first time that Ghr can decrease memory retention in a non-mammalian species, suggesting that Ghr may play an important role in the processes of memory retention in birds.

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 [3 H]-flunitrazepam receptors in synaptosomal membranes from forebrain. In addition, low sub-solubilizing 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 [3 H]-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.

Preparation of a three-dimensional extracellular matrix by decellularization of rabbit livers

INTRODUCTION the availability of transplantable livers is not sufficient to fulfill the current demand for grafts, with the search for therapeutic alternatives having generated different lines of research, one of which is the use of decellularized three-dimensional biological matrices and subsequent cell seeding to obtain a functional organ. OBJECTIVE to produce a decellularization protocol from rabbit liver to generate a three-dimensional matrix. METHODS a combination of physical, chemical (Triton X-100 and SDS) and enzymatic agents to decellularize rabbit livers was used. After 68 h of retrograde perfusion, a decellularized translucent matrix was generated. To evaluate if the decellularization protocol was successful, with the extracellular matrix being preserved, we carried out histological (light microscopy and scanning electron microscopy) and biochemical (DNA quantification) studies. RESULTS the decellularization process was verified by macroscopic observation of the organ using macroscopic staining, which revealed a correct conservation of bile and vascular trees. A microscopic observation corroborated these macroscopic results, with the hematoxylin-eosin staining showing no cells or nuclear material and the presence of a portal triad. Wilde´s staining demonstrated the conservationof reticulin fibers in the decellularized matrix. In addition, scanning electron microscopy revealed a preserved Glisson´s capsule and a decellularized matrix, with the DNA quantification being less than 10 % in the decellularized liver compared to control. Finally, the time taken to develop the decellularization protocol was less than 96 hours. CONCLUSIONS the proposed decellularization protocol was correct, and was verified by an absence of cells. The hepatic matrix had preserved vascular and bile ducts with a suitable three-dimensional architecture permitting further cell seeding.

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.

Chick imprinting performance and susceptability 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.

Acute stress or systemic insulin injection increases flunitrazepam sensitive-GABAA receptor density in synaptosomes of chick forebrain: Modulation by systemic epinephrine

Interactions between acute stress and systemic insulin and epinephrine on GABAA receptor density in the forebrain were studied. Here, 10 day-old chicks were intraperitoneally injected with insulin, epinephrine or vehicle and then immediately stressed by partial water immersion for 15 min and killed by decapitation. Non-stressed controls were similarly injected, then returned to their rearing boxes for 15 min and then killed. Forebrains were dissected and GABAA receptor density was measured ex vivo in synaptosomes by 3[H]-flunitrazepam binding assay. In non-stressed chicks, insulin at 1.25, 2.50 and 5.00 IU/kg of body weight (non-hypoglycemic doses) increased Bmax by 33, 53 and 44% compared to saline, respectively. A similar increase of 41% was observed in receptor density after stress. However, the insulin effect was not additive to the stress-induced increase suggesting that both effects occur through similar mechanisms. In contrast, epinephrine, at 0.25 and 0.5 mg/kg did not induce any changes in Bmax in non-stressed chicks. Nevertheless, after stress these doses increased the receptor density by about 13 and 27%, respectively. Similarly, the same epinephrine doses co-administered with insulin (2.50 IU/kg), increased the receptor density by about 20% compared to insulin alone. These results suggest that systemic epinephrine, perhaps by evoking central norepinephrine release, modulates the increase in forebrain GABAA receptor binding induced by both insulin and stress.

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.

α-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.

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.

Central α- and β-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 β-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. β-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.