Effect of GABA receptor agonist phenibut on behavior and respiration of rabbits in the negative emotional situation

The influence of systemic injection of GABA-receptor agonist--phenibut (40 mg/kg, s/c) on open field behavior, behavioral reactivity and changes in respiratory parameters after exposure of negative emotional stimuli was studied in three rabbit groups differentiated by locomotor activity in open field (active, passive and medium-active animals). The injection of phenibut results in decrease of rabbits horizontal locomotor activity and some components of research behavior in open field and also decrease of behavioral reactivity on emotional stimuli. At the same time the probability of both an active orienting exploratory or defensive reactions and passive reactions (freezing) were decreased. The influence of phenibut depended on typological features of rabbits: the most potent effect occurred upon behavior of active rabbits, less on passive animals and practically none on medium-active rabbits. The phenibut injection results in increase of duration of inhalation during exposure to emotional stimuli, whereas it decreased in normal.     

Emotional reactivity of rats and their progeny after hormonal modification of intrauterine development

Emotional reactivity of two rat generations was estimated in open field test and by the corticosteroids level under motor restriction after hydrocortisone injections (50 mg/kg) to their mothers (the 1st generation) or grandmothers (the 2nd generation) on 16 and 18 days of gestation. The 3-, 6- or 12-month-old males and females of the 1st generation had decreased emotional reactivity. On the contrary, the animals of the 2nd generation had increased defecation, but decreased ambulation scores in the open field test, that characterised them as more emotional in comparison to the control rats. Thus, the corticosteroids can be a factor of "nongenetic transmission of information" in the development of animals' emotional reactivity mechanisms.     

Japanese Quail’s Genetic Background Modulates Effects of Chronic Stress on Emotional Reactivity but Not Spatial Learning

Chronic stress is known to enhance mammals’ emotional reactivity and alters several of their cognitive functions, especially spatial learning. Few studies have investigated such effects in birds. We investigated the impact of a two-week stress on Japanese quail’s emotional reactivity and spatial learning. Quail is an avian model widely used in laboratory studies and for extrapolation of data to other poultry species. As sensitivity to chronic stress can be modulated by intrinsic factors, we tested juvenile female Japanese quail from three lines, two of them divergently selected on tonic immobility duration, an indicator of general fearfulness. The different emotional reactivity levels of quail belonging to these lines can be revealed by a large variety of tests. Half of the birds were submitted to repeated unpredictable aversive events for two weeks, whereas the other half were left undisturbed. After this procedure, two tests (open field and emergence tests) evaluated the emotional reactivity of treated and control quails. They were then trained in a T-maze for seven days and their spatial learning was tested. The chronic stress protocol had an impact on resting, preening and foraging in the home cage. As predicted, the emotional reactivity of treated quails, especially those selected for long tonic immobility duration, was higher. Our spatial learning data showed that the treatment enhanced acquisition but not memorization. However, intrinsic fearfulness did not seem to interact with the treatment in this test. According to an inverted U-shaped relationship between stress and cognition, chronic stress can improve the adaptability of birds to a stressful environment. We discussed the mechanisms possibly implied in the increase of emotional reactivity and spatial abilities.     

Effect of a 3-hydroxypyridine-class antioxidant on the functional activity of the central nervous system in mice of different age groups

Characteristics of emotional reactivity, orienting-investigating behaviour and of preservation of conditioned reflex of passive avoidance were used for estimation of the functional activity of animals CNS and also of psychotropic effect of antioxidant 2-ethyl-6-methyl-3-oxypyridine (3-OP) on mice of different age. Studies were carried out on white outbred young (3 months) and elderly (15 months) male mice. Different character was observed of the emotional reactions of different age mice to aversive stimuli and to the open field conditions. Differences were noted of psychotropic effects of 3-OP in 3 and 15-months animals.     

Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5′-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues.     

A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.     

Cysteine pKa depression by a protonated glutamic acid in human DJ-1

Human DJ-1, a disease-associated protein that protects cells from oxidative stress, contains an oxidation-sensitive cysteine (C106) that is essential for its cytoprotective activity. The origin of C106 reactivity is obscure, due in part to the absence of an experimentally determined p K a value for this residue. We have used atomic-resolution X-ray crystallography and UV spectroscopy to show that C106 has a depressed p K a of 5.4 +/- 0.1 and that the C106 thiolate accepts a hydrogen bond from a protonated glutamic acid side chain (E18). X-ray crystal structures and cysteine p K a analysis of several site-directed substitutions at residue 18 demonstrate that the protonated carboxylic acid side chain of E18 is required for the maximal stabilization of the C106 thiolate. A nearby arginine residue (R48) participates in a guanidinium stacking interaction with R28 from the other monomer in the DJ-1 dimer and elevates the p K a of C106 by binding an anion that electrostatically suppresses thiol ionization. Our results show that the ionizable residues (E18, R48, and R28) surrounding C106 affect its p K a in a way that is contrary to expectations based on the typical ionization behavior of glutamic acid and arginine. Lastly, a search of the Protein Data Bank (PDB) produces several candidate hydrogen-bonded aspartic/glutamic acid-cysteine interactions, which we propose are particularly common in the DJ-1 superfamily.     

Compensation in behavioral disorders in rats receiving 5,7-DHT neonatally and by transplantation of embryonic raphe nucleus tissue

In 62 male Wistar rats the influence was studied of the transplanted embryonal tissue of raphe nuclei (NR) on the mechanisms of compensation of disturbances of exploratory activity, sensory attention, learning and emotional reactivity induced by neonatal injection of 5,7-DHT. In histochemical studies by Falk-Hillarp method the presence of yellow fluorescence confirmed the specificity of transplanted 5-HT neurones. It is found that NR transplantation causes in animals after 3 months recovery of orienting reaction to sensory stimuli, reduces rats reactivity in the open field, restores the ability to discrimination of emotionally positive influence, disturbed by neonatal injection of 5,7-DHT. The obtained data show the possibility of compensation of behaviour disturbances caused by chronic deprivation of 5-HT system activity by transplantation in the neocortex parenchyma of the embryonal tissue, containing serotoninergic neurones.     

A comparative analysis of learning and exploratory behavior in rats with different resistances to stress exposures and to the brain monoamine level]

Wistar rats stable (S) to sound stimulus differed from the unstable (NS) ones by a heightened investigatory activity in condition of moderate stress in the open field test, by heightened reactivity to sensory stimuli of various modalities (somatosensory, visual and olfactory), lowered level of investigatory behaviour in the test of burrow chamber. S-rats differed from NS-animals by a higher ability to learning of goal-directed reaction and a lower ability to discrimination of different emotional influences. The results of biochemical analysis of the content of biogenic amines in various brain structures revealed in stable rats an increase of noradrenaline level and in non-stable ones--a higher level of dopamine and serotonin.     

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS

A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [³H]DA and inhibitor [³H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [³H]DA and [³H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.     

Apha-tocopherol in a complex with dimethyl sulfoxide--an agent possessing highly effective adaptogenic action in chronic emotional-pain stress in rats

Alpha-tocopherol (5 mg/kg) administered perorally with dimethyl sulfoxide (50 mg/kg) in chronic emotional pain stress in rats possesses an effective antistress action, exceeding the effects of these drugs administered separately. Their prophylactic complex administration prevents the hypertension produced by stress, disturbance of reactivity of the vegetative nervous system during functional load, change of the behaviour in the open field. Adaptogenic action of the drugs is accompanied by a reduction of the content of free-radical oxidation products and by raising of superoxide scavenging activity in the brain and blood serum, by raising of phospholipids content, lowering of cholesterol content and of the ratio of cholesterol phospholipids in the brain extracts.     

Pleiotropic effect of a locus on chromosome 4 influencing alcohol drinking and emotional reactivity in rats

A QTL search in a segregating F2 intercross between HEP (High-Ethanol Preferring line) and wistar-kyoto (WKY, a low-alcohol consuming strain) rats identified a locus on chromosome 4 linked to the consumption of a 5% alcohol solution offered as a free choice with water (Terenina-Rigaldie et al. submitted). In order to confirm and analyse the influence of this locus, F2 rats were selected according to their genotype at the markers flanking the QTL and bred in order to obtain two groups of rats homozygous HEP/HEP ('HIGH' line) or WKY/WKY ('LOW' line) at the QTL, the rest of the genome being randomly inherited from one or the other founder strain. These two groups of animals displayed large differences in emotional reactivity (open field, elevated-plus maze), sensitivity to taste reinforcers (saccharin, quinine) and alcohol consumption (either forced or as a free choice with water). These results confirm the influence of this locus on alcohol intake and emotional reactivity traits, and suggest a pleiotropic effect of the gene(s) involved. Current research aims at the identification of this (these) gene(s).     

State-dependent chemical reactivity of an engineered cysteine reveals conformational changes in the outer vestibule of the cystic fibrosis transmembrane conductance regulator

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET+. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.     

Influence of emotional stimuli on behavior and respiration of rabbits with various locomotor activity in the "open field"

Different behavioral reactivity of rabbit groups differentiated by locomotor activity in the "open field" was revealed during exposure to emotional stimuli (rustle, loud sound, pressuring on back of the neck, vibroacoustic tactile stimulation of an ear). In passive rabbits, the active locomotor reactions were induced harder and freezing was obtained easier than in active animals. During exposure to sound stimuli, passive rabbits increased their locomotion more rarely than active animals, pressing on back of the neck produced longer freezing, a threshold of defensive ear shaking in response to a vibroacoustic stimulus in passive animals was highest. Training to mild immobilization increased the threshold of defensive responses in active rabbits and animals of the intermediate type. Changes in respiratory parameters were correlated with behavioral reactions to emotional stimuli. The duration of exhalation and respiratory cycle increased during freezing and increased during enhanced locomotion. The duration of inhalation decreased in response to emotional stimuli irrespective of a behavioral reaction. The respiratory reactions to emotional stimuli differed in rabbits of different groups. The respiratory rate more frequently changed in passive rabbits than in animals of other groups. Passive animals reacted mainly by exhalation, active rabbits and animals from the intermediate group predominantly responded by inhalation.     

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

Multiple transmembrane (TM) segments line the pore of the cystic fibrosis transmembrane conductance regulator Cl(-) channel; however, the relative alignment of these TMs and their relative movements during channel gating are unknown. To gain three-dimensional structural information on the outer pore, we have used patch clamp recording to study the proximity of pairs of cysteine side chains introduced into TMs 6 and 11, using both disulfide cross-linking and Cd(2+) coordination. Following channel activation, disulfide bonds could apparently be formed between three cysteine pairs (of 15 studied): R334C/T1122C, R334C/G1127C, and T338C/S1118C. To examine the state dependence of cross-linking, we combined these cysteine mutations with a nucleotide-binding domain mutation (E1371Q) that stabilizes the channel open state. Investigation of the effects of the E1371Q mutation on disulfide bond formation and Cd(2+) coordination suggests that although R334C/T1122C and T338C/S1118C are closer together in the channel open state, R334C/G1127C are close together and can form disulfide bonds only when the channel is closed. These results provide important new information on the three-dimensional structure of the outer mouth of the cystic fibrosis transmembrane conductance regulator channel pore: TMs 6 and 11 are close enough together to form disulfide bonds in both open and closed channels. Moreover, the altered relative locations of residues in open and in closed channels that we infer allow us to propose that channel opening and closing may be associated with a relative translational movement of TMs 6 and 11, with TM6 moving "down" (toward the cytoplasm) during channel opening.     

Comparative study of the psychotropic activity of tuftsin and its analogs

Tuftsin and its Leu1 and D-Arg4 analogs displayed stimulating activity in experimental behavioral despair in mice. In rats with different types of emotional reactions and with destroyed catecholamine terminals (6-OHDA treatment), tuftsin increased exploratory activity, with fear manifestations being decreased and avoidance behavior improved. This was shown while testing the rats in the "open field" and according to the ability to accomplish an extrapolation task of avoiding critical stress-situation. Leu1-tuftsin increased the emotional stress and sharply hindered the avoidance reaction, while D-Arg4-tuftsin modulated the behavior of the animals with increased emotional reactivity and made the avoidance behavior prompter. Pentapeptide, an inhibitor of tuftsin stimulation of phagocytosis, had no significant effect on the behavior. Modifications in the structure of tuftsin resulted both in the changes in phagocytosis-stimulating activity and the appearance of other psychotropic effects.     

Participation of some meteorological factors on the determination of emotional reactivity

Summary Rats were exposed to conditions of various temperature (21, 27 and 33°C) and air humidity (50%, 70% and 90%) and their emotional reactivity was estimated.Increased temperature and air humidity increases more or less expressively the emotional reactivity. It was further stated that interindividual differences of emotional behaviour are maintained in conditions of lower levels of variously warm and humid air.     

Cyclic nucleotide levels in the brain of inbred mice in emotional stress exposure

The content of cAMP and cGMP in different brain regions was studied in C57Bl/6 and BALB/c mice at rest and upon exposure to emotional stress induced by open field technique. Interstrain differences in baseline nucleotide content, differences in nucleotide distribution in the brain regions and changes in their concentration after stress have been revealed.     

Effects of maternal deprivation on adrenal and behavioural responses in rats with anterodorsal thalami nuclei lesions

There is evidence that repeated maternal isolation of neonatal rats may influence both emotional behavior and Hypothalamic-Pituitary Adrenal (HPA) activity. On the other hand the Anterodorsal Thalami Nuclei (ADTN) exerts an inhibitory influence on the hypophyso-adrenal system under basal and stressful conditions. In the present work we investigated whether neonatal maternal deprivation produces long term effects on the ADTN regulation of behavioral patterns (open field test) and on HPA axis activity. Specifically, we sought to determine whether adult female rats with ADTN lesions, previously isolated for 4.5 hours daily during the first 3 weeks of life, react in endocrinologically and behaviourally distinct manner as compared to controls. The examined groups were: non maternally deprived (NMD)/sham lesioned, NMD/lesioned, maternally deprived (MD)/sham lesioned, MD/lesioned with and without the open field test. At 3 months MD/sham lesioned animals showed a marked decrease in ambulation (P < 0.01), and with ADTN lesion, the rearing values were lower (P < 0.01) and grooming higher (P < 0.05) than NMD. This last data would indicate a high emotional index. Regarding the activity of the HPA axis, maternal deprivation induced a significant decrease in plasma ACTH concentration both in sham and lesioned animals (P < 0.001), and plasma Corticosterone (C) increased in sham animals (P < 0.001). This data would indicate a higher sensitivity of the adrenal glands. After the open field test ACTH and C were different between deprived and non-deprived animals depending on the ADTN lesion. Taking into consideration the increase of ACTH levels in sham lesioned MD animals exposed to the test, we could conclude that this new situation was a stressful situation. Finally in the present work, it was very difficult to relate the behavioral parameters with the endocrine data. It is known that depending on the context, corticosteroids may produce opposite effects on emotional behavior via different receptors in the brain.In summary, neonatal maternal deprivation induced alterations of behavioral patterns and affected the ADTN inhibitory influence on ACTH and C secretion.