Differential expression of hypothalamic fear- and stress-related genes in broiler chickens showing short or long tonic immobility.

The serotonin system and the hypothalamic-pituitary-adrenal axis play important roles in modulating fear and stress-coping characteristics. Tonic immobility (TI) is a fear-related phenotype, and previously we have shown that broiler chickens showing short TI (STI) duration experience better growth performance and higher adaptability to stress. Here, we sought to further elucidate the central mechanisms underlying the phenotypic differences between chickens showing STI and long TI duration, by comparing the hypothalamic expression of genes in the serotonergic system and the hypothalamic-pituitary-adrenal axis under basal and corticosterone-exposed situations. The STI broilers had significantly lower (P < 0.01) hypothalamic expression of serotonin reuptake transporter and serotonin receptor 1A. Moreover, 11ß-hydroxysteroid dehydrogenase type 2 was expressed significantly lower in STI chickens at the level of both mRNA (P < 0.01) and protein (P < 0.05). Hypothalamic expression of glucocorticoid receptor (GR) mRNA tended to be higher (P < 0.059) in long TI chickens, but the protein content was approximately 2 times higher (P < 0.01) in STI chickens. The uncoupled expression of GR mRNA and protein was associated with significantly lower (P < 0.05) expression of gga-miR-181a, gga-miR-211, and gga-miR-22, which are predicted to target GR, in STI chickens. Corticosterone administration reduced the mRNA expression of postsynaptic serotonin receptors, 5-hydroxytryptamine receptor 1B (P = 0.059) and 5-hydroxytryptamine receptor 7 (P < 0.05), yet significantly increased the protein content of 11ß-hydroxysteroid dehydrogenase type 2 (P < 0.05). These results suggest that broilers of different TI phenotypes have a distinct pattern of hypothalamic expression of fear- and stress-related genes.

Is there tonic immobility in humans? Biological evidence from victims of traumatic stress.

Tonic immobility, characterized by profound motor inhibition, is elicited under inescapable threat in many species. To fully support the existence of tonic immobility in humans, our aim was to elicit this reaction in a laboratory setting and measure it objectively. To mimic exposure to life-threatening events in the lab, trauma-exposed participants with PTSD (n=18) and without PTSD (n=15) listened to the script of their autobiographical trauma. Posturography and electrocardiography were employed. Reports of script-induced immobility were associated with restricted area of body sway and were correlated with accelerated heart rate and diminished heart rate variability, implying that tonic immobility is preserved in humans as an involuntary defensive strategy. Immobility reports seemed more evident in PTSD, suggesting that, in some patients, tonic immobility may be elicited during re-experiencing episodes in daily life. This study provided a measure of tonic immobility, a peritraumatic reaction for which cumulative clinical evidence had linked to the severity of PTSD.

Dietary arginine depletion reduces depressive-like responses in male, but not female, mice.

Previous behavioral studies have manipulated nitric oxide (NO) production either by pharmacological inhibition of its synthetic enzyme, nitric oxide synthase (NOS), or by deletion of the genes that code for NOS. However manipulation of dietary intake of the NO precursor, L-arginine, has been understudied in regard to behavioral regulation. L-Arginine is a common amino acid present in many mammalian diets and is essential during development. In the brain L-arginine is converted into NO and citrulline by the enzyme, neuronal NOS (nNOS). In Experiment 1, paired mice were fed a diet comprised either of an L-arginine-depleted, L-arginine-supplemented, or standard level of L-arginine during pregnancy. Offspring were continuously fed the same diets and were tested in adulthood in elevated plus maze, forced swim, and resident-intruder aggression tests. L-Arginine depletion reduced depressive-like responses in male, but not female, mice and failed to significantly alter anxiety-like or aggressive behaviors. Arginine depletion throughout life reduced body mass overall and eliminated the sex difference in body mass. Additionally, arginine depletion significantly increased corticosterone concentrations, which negatively correlated with time spent floating. In Experiment 2, adult mice were fed arginine-defined diets two weeks prior to and during behavioral testing, and again tested in the aforementioned tests. Arginine depletion reduced depressive-like responses in the forced swim test, but did not alter behavior in the elevated plus maze or the resident intruder aggression test. Corticosterone concentrations were not altered by arginine diet manipulation in adulthood. These results indicate that arginine depletion throughout development, as well as during a discrete period during adulthood ameliorates depressive-like responses. These results may yield new insights into the etiology and sex differences of depression.

Neuropeptide S receptor deficiency modulates spontaneous locomotor activity and the acoustic startle response.

The present study investigated the phenotype of heterozygous and homozygous neuropeptide S receptor (Npsr) deficient C57BL/6 mice in NPS- and cocaine induced hyperactivity, spontaneous and reactive locomotor activity, elevated plus maze, conditioned fear, and prepulse inhibition of the acoustic startle response. In Npsr-deficient mice, a strong reduction of spontaneous locomotor activity and of the startle magnitude was observed; heterozygous mice had an intermediate phenotype. In the other experiments, Npsr deficiency leads to no or only a very modest phenotype. These results support an important role of neuropeptide S in regulating locomotor activity.

Effect of heat and several additives related to stress levels on fluctuating asymmetry, heterophil:lymphocyte ratio, and tonic immobility duration in White Leghorn chicks.

The purpose of this study was to analyze the effects of heat and several additives related to stress on fluctuating asymmetry (groups 1 to 10), heterophil:lymphocyte ratio (groups 1 to 3 and 8 to 10), and tonic immobility duration (groups 1 to 7 and 10) in White Leghorn chicks at 42 d of age. Chicks in group 1 (heat) were reared with temperatures 8°C greater than those of the control group. Groups 2 to 9 consisted of chicks reared with temperatures 8°C greater than those of the control group and addition of capsaicin, allicin, ascorbic acid, tryptophan, brewer's yeast, lactic acid, corticosterone, or cholesterol in diet. Chicks in group 10 (control) were reared with standard temperatures. Heat effect was significant (P<0.05) for the heterophil:lymphocyte ratio, which was greater in heat-stressed chicks without any additives and smaller in control chicks. There were no significant differences for the fluctuating asymmetry and the tonic immobility duration between both groups. Heterophil:lymphocyte ratio for heat-stressed chicks with capsaicin or allicin was significantly lower (P<0.05) than that of heat-stressed chicks without any additives. Capsaicin effect was not significant for the fluctuating asymmetry and the tonic immobility duration, whereas allicin significantly increased fluctuating asymmetry of wing length and tonic immobility duration (P<0.05). The addition of lactic acid or corticosterone resulted in greater fluctuating asymmetry of wing length of heat-stressed chicks (P<0.05). In conclusion, an increased heterophil:lymphocyte ratio was found in heat-stressed chicks without additives, indicating that it is a more reliable indicator of the effect of heat in chicks. In addition, dietary capsaicin or allicin supplementation was effective to alleviate the stress induced by the high temperature, as indicated by a lower heterophil:lymphocyte ratio.

Correlations between neuron activity in the sensorimotor cortex of the right and left hemispheres in rabbits during a defensive dominant and "animal hypnosis".

A latent focus of excitation with a rhythmic nature (a defensive dominant focus) was created in the CNS of rabbits. The focus was formed by threshold electrocutaneous stimulation of the left forelimb using series of impulses consisting of 15-20 stimuli with interstimulus intervals of 2 sec. The linked activity of cells in the sensorimotor cortex of the right and left hemispheres was analyzed. When cross-correlation histograms of the spike activity of sensorimotor cortex neurons in the left hemisphere were constructed and analyzed in relation to spikes of high and intermediate amplitude recorded in the right hemisphere, the linked activity of 15% and 23% of neuron pairs, respectively, showed predominance of a rhythm equal or close to the stimulation rhythm used to form the dominant focus. When the appearance times of spikes from neurons in the sensorimotor cortex of the right hemisphere were analyzed in relation to spikes of high and intermediate amplitude recorded in the cortex of the left hemisphere, predominance of 2-sec rhythms was seen in the linked activity of only 3% and 10% of neuron pairs, respectively. After induction of "animal hypnosis," differences between the hemispheres in relation to this measure leveled out.

Dynamics of changes in electrical activity in the rabbit cerebral cortex during sequential sessions of "animal hypnosis".

The dynamics of changes in individual electrical activity rhythms in the premotor, sensorimotor, and temporal-parietal areas of the cortex in both hemispheres were studied in chronic experiments in rabbits during sequential sessions of "animal hypnosis." These experiments showed that during the first session of "animal hypnosis," significant changes in electrical activity occurred only in the premotor area of the cortex of the right hemisphere, where there were increases in spectral power in the delta-1 and delta-2 ranges and decreases in spectral power in other ranges of electrical activity. Subsequent sessions of "animal hypnosis" formed increasing changes in electrical activity, which were particularly marked in cortical areas in the right hemisphere. Significant changes in spectral power in the delta and theta ranges of electrical activity in cortical areas did not arise at the beginning of the hypnotic state, but after 4-6 min. During the third session of "animal hypnosis," the course of electrical activity in the alpha and beta rhythms in the premotor and sensorimotor areas of the cortex became wave-like in nature.

Depression-like behavior and reduced plasma testosterone levels in the senescence-accelerated mouse.

During aging, levels of testosterone gradually decline in men and low levels of testosterone in aged men are accompanied by increased incidence of depressive disorders. The senescence-accelerated-prone mouse 10 (SAMP10) is well known as an animal model of aging. The purpose of this study was to investigate the motor function, anxiety levels, depression-related emotional responses, attentional function and plasma levels of testosterone and dehydroepiandrosterone (DHEA) in SAMP10. SAMP10 exhibited a significant prolongation of immobility time compared to that of the aged-matched control senescence-accelerated-resistant mouse 1 (SAMR1) in the tail suspension test for measuring depression. Moreover, significant low levels of plasma testosterone but not DHEA were found in SAMP10, and the testosterone levels were inversely correlated with the depression-like behavior. By contrast, we did not observe any significant differences between SAMP10 and SAMR1 in the open-field, rota-rod, elevated plus-maze, marble-burying behavior, or prepulse inhibition test. The results of the present study indicate that testosterone may play an important role in the depression-like behavior in SAMP10.

Behavioural phenotype of APPC100.V717F transgenic mice over-expressing a mutant Abeta-bearing fragment is associated with reduced NMDA receptor density.

The aim of this study was to characterize APPC100.V717F transgenic (TgC100.V717F) mice which over-express a mutant C100 fragment of the amyloid precursor protein. The mice were compared to TgC100 wild type mice (TgC100.WT) and non-transgenic controls at 4-9 and 16-22 months of age. TgC100.V717F mice showed behavioural hyperactivity, particularly at a younger age, as shown by increased numbers of elevated plus maze arm entries and Y-maze arm entries, enhanced baseline locomotor activity in the open field, and enhanced amphetamine-induced hyperlocomotion. This hyperactivity was less pronounced in TgC100.WT which only displayed significant differences to non-transgenic controls at a younger age for the number of Y-maze arm entries and baseline locomotor activity in the open field. In addition, TgC100.V717F mice, but not TgC100.WT, demonstrated cognitive deficits, as shown by reduced spontaneous alternation in the Y-maze and markedly reduced retention in a passive avoidance test. At an older age, TgC100.V717F mice showed enhanced startle and increased immobility time in the forced swim test. In the TgC100.V717F mice, but not TgC100.WT, the behavioural changes were paralleled by a significant reduction in the expression of hippocampal NMDA receptor subunits types 1 and 2A. Concomitantly, we detected axonal disruption and apoptosis in the hippocampus of TgC100.V717F mice. In conclusion, these data demonstrate that the mutant C100 fragment is an effector of biochemical and both cognitive and non-cognitive behaviours. These transgenic mice may be a model for the psychotic features associated with early Alzheimer's disease.

[Correlations of activity of neurons of sensorimotor cortex of the right and left brain hemispheres of rabbits during defensive dominant and "animal hypnosis"].

A hidden excitation focus of the rhythmic nature (a rhythmic defensive dominant focus) was produced in the rabbit's CNS. The focus was formed by means of threshold electrodermal stimulation of the left forelimb by series of pulses consisting of 15-20 stimuli with 2 s intervals between the pulses. Correlated activity of cells in the sensorimotor cortex of the right and left brain hemispheres was analyzed. In cases when crosscorrelation histograms were constructed by the results of the analysis of discharges of the left-side cortical of neurons regarding high- and middle-amplitude pulses in a right hemisphere, 15 and 23 % of correlated neural pairs, respectively, revealed the prevalence of the rhythm identical or close to the initial rhythm of stimulation that formed the hidden excitation focus. In contrast, in cases when the same analysis was applied to the right-side cortical neurons regarding high- and middle-amplitude discharges in the left hemisphere, prevalence of the dominant 2-second rhythm was revealed in correlated activity of only 3 and 10% of neural pairs, respectively. After the exposure to "animal hypnosis" procedure, the distinctions between the brain in this parameter were eliminated.

[The dynamics of modification of electrical activity of the rabbit brain during consistent "animal hypnosis" series].

The dynamics of modification of individual rhythms of electrical activity in both hemispheres was studied under condition of chronic experiments in rabbits during "animal hypnosis" sessions. It was shown that, after the first "animal hypnosis" session, significant changes in the spectral power of electrical activity appeared only in the right premotor cortical area. They consisted in an increase in the spectral power of the delta1 and delta2 rhythms and a decrease in the spectral power of the other rhythms. During the next "animal hypnosis" sessions, changes in the electrical activity became stronger, especially in the right hemisphere. Significant changes in the spectral power of the delta and theta rhythms appeared not at the beginning of the "animal hypnosis" session but 4-6 min later. During the third "animal hypnosis" session, the power of the alpha and beta rhythms in the premotor and sensorimotor cortical areas of both hemispheres varied in an undulatory way.

Glycine receptors contribute to hypnosis induced by ethanol.

BACKGROUND: Glycine is a major inhibitory neurotransmitter in the adult central nervous system (CNS), and its receptors (GlyRs) are well known for their effects in the spinal cord and the lower brainstem. Accumulating evidence indicates that GlyRs are more widely distributed in the CNS, including many supraspinal regions. Previous in vitro studies have demonstrated that ethanol potentiates the function of these brain GlyRs, yet the behavioral role of the brain GlyRs has not been well explored. METHODS: Experiments were conducted in rats. The loss of righting reflex (LORR) was used as a marker of the hypnotic state. We compared the LORR induced by systematic administration of ethanol and of ketamine in the absence and presence of the selective glycine receptor antagonist strychnine. Ketamine is a general anesthetic that does not affect GlyRs. RESULTS: Systemically administered (by intraperitoneal injection) ethanol and ketamine dose-dependently induced LORR in rats. Furthermore, systemically administered (by subcutaneous injection) strychnine dose-dependently reduced the percentage of rats exhibiting LORR induced by ethanol, increased the onset time, and decreased the duration of LORR. Strychnine had no effect, however, on the LORR induced by ketamine. CONCLUSIONS: Given that hypnosis is caused by neuronal depression in upper brain areas, we therefore conclude that brain GlyRs contribute at least in part to the hypnosis induced by ethanol.

An animal model of hypnotic pain attenuation.

The tonic immobility state (TIS) in rabbit has great potential as an animal model of hypnosis in humans, due to the strong similarities between TIS and hypnosis. It is additionally the only mammalian model available for studying mechanisms of hypnosis. However, its ability to modulate pain perception has yet to be clearly determined. The present experiment examined the effect of nociceptive conditioning in normal, TIS and lidocaine-treated rabbits. This involved three separate phases. In phase one all animals were trained on a continuous performance test (CPT). In the second phase animals were given Pavlovian conditioning to an auditory CS paired with a nociceptive US. In the third phase the animals are given a sensory recognition test. The control group were conditioned during a normal state, the TIS group were conditioned during TIS, the pain control animals were conditioned after lidocaine injection in the skin area stimulated by the nociceptive US. All animals in the normal group showed the greatest amount of pain conditioning, with an average disruption time (dt) of 175s. Animals in the lidocaine group showed little signs of conditioning with an average dt of 16s. Animals conditioned during TIS had an average dt of 42s. These results show that TIS can modulate pain perception similar to animals that did not experience a pain CS. They additionally argue that tonic immobility can produce attenuation of pain perception similar to hypnosis in humans.

Gamma-aminobutyric acid-mediated neurotransmission in the pontine reticular formation modulates hypnosis, immobility, and breathing during isoflurane anesthesia.

BACKGROUND: Many general anesthetics are thought to produce a loss of wakefulness, in part, by enhancing gamma-aminobutyric acid (GABA) neurotransmission. However, GABAergic neurotransmission in the pontine reticular formation promotes wakefulness. This study tested the hypotheses that (1) relative to wakefulness, isoflurane decreases GABA levels in the pontine reticular formation; and (2) pontine reticular formation administration of drugs that increase or decrease GABA levels increases or decreases, respectively, isoflurane induction time. METHODS: To test hypothesis 1, cats (n = 5) received a craniotomy and permanent electrodes for recording the electroencephalogram and electromyogram. Dialysis samples were collected from the pontine reticular formation during isoflurane anesthesia and wakefulness. GABA levels were quantified using high-performance liquid chromatography. For hypothesis 2, rats (n = 10) were implanted with a guide cannula aimed for the pontine reticular formation. Each rat received microinjections of Ringer's (vehicle control), the GABA uptake inhibitor nipecotic acid, and the GABA synthesis inhibitor 3-mercaptopropionic acid. Rats were then anesthetized with isoflurane, and induction time was quantified as loss of righting reflex. Breathing rate was also measured. RESULTS: Relative to wakefulness, GABA levels were significantly decreased by isoflurane. Increased power in the electroencephalogram and decreased activity in the electromyogram caused by isoflurane covaried with pontine reticular formation GABA levels. Nipecotic acid and 3-mercaptopropionic acid significantly increased and decreased, respectively, isoflurane induction time. Nipecotic acid also increased breathing rate. CONCLUSION: Decreasing pontine reticular formation GABA levels comprises one mechanism by which isoflurane causes loss of consciousness, altered cortical excitability, muscular hypotonia, and decreased respiratory rate.

Effects of "animal hypnosis" on a rhythmic defensive dominant.

A defensive dominant was created in rabbits using rhythmic electrocutaneous stimulation of the left forelimb at a frequency of 0.5 Hz. After stimulation ended, the latent excitation state was tested using sound stimuli. Animals responded either with increases in non-rhythmic paw muscle activity or with rhythmic twitching of the paw at a frequency close to that of the electrocutaneous stimulation. After hypnotization, the incidence of rhythmic responses to the stimulation testing the dominant focus increased, while the incidence of non-rhythmic responses decreased.

[Effect of "animal hypnosis" on the rhythmic defensive dominanta].

A defensive dominanta (stationary excitation focus) in the sensorimotor cortex of rabbits was formed by rhythmical electrodermal paw stimulation with the frequency of 0.5 Hz. After cessation of the stimulation, the state of hidden excitation was tested with acoustic stimuli, in response to which nonrhythmic activity of leg muscles increased or the leg rhythmically startled with the frequency close to that of the electrodermal stimulation. After conducting a routine hypnotizing procedure, the incidence of the rhythmic responses to testing stimulation increased, while the incidence of nonrhythmic responses decreased.

KCC2-deficient mice show reduced sensitivity to diazepam, but normal alcohol-induced motor impairment, gaboxadol-induced sedation, and neurosteroid-induced hypnosis.

GABA(A) receptors mediate both fast phasic inhibitory postsynaptic potentials and slower tonic extrasynaptic inhibition. Hyperpolarizing phasic GABAergic inhibition requires the activity of neuron-specific chloride-extruding potassium-chloride cotransporter KCC2 in adult CNS. However, the possible role of KCC2 in tonic GABAergic inhibition and the associated behaviors is unknown. Here, we have examined the role of KCC2 in phasic vs tonic GABA inhibition by measuring the behavioral effects of pharmacological agents that presumably enhance phasic vs tonic inhibition in mice that retain 15-20% of normal KCC2 protein levels. These KCC2-deficient mice show decreased sensitivity to diazepam-induced sedation and motor impairment consistent with the reported role for KCC2 in fast hyperpolarizing inhibition. In contrast, the mice exhibit normal responses to low-dose alcohol-induced motor impairment, gaboxadol-induced sedation, and neurosteroid-induced hypnosis; behaviors thought to be associated with tonic GABAergic inhibition. Electrophysiological recordings show that the tonic conductance is not affected. The results suggest that KCC2 activity is more critical for behaviors dependent on phasic than tonic GABAergic inhibition.

Emotional reactivity modulates autonomic responses to an acoustic challenge in quail.

Emotional reactivity modulates autonomic responses to an acoustic challenge in quail. Physio Behav 00(0) 000-000, 2006. This study investigated the relationship between emotional reactivity and behavioral and autonomic responses to an acoustic stimulus in quail. It was hypothesized that birds with high emotional reactivity would have higher motor inhibition combined with higher sympathetic activation than birds with low emotional reactivity. Two experiments were performed. The first looked for correlations between emotional reactivity, evaluated by a tonic immobility test, and motor and Heart Rate Variability in relation to an acoustic stimulus. The second experiment compared the motor and autonomic responses to the acoustic stimulus of quail selected on either long (LTI) or short (STI) duration of tonic immobility. The first experiment showed that the acoustic stimulation induced motor inhibition and cardiac activation. Correlations were found between tonic immobility duration and both autonomic activity before stimulation and sympathovagal balance after stimulation. In the second experiment, LTI quail showed strong sympathetic activation, whereas STI quail showed parasympathetic and sympathetic activation. The activation of the parasympathetic system induced by the noise in STI quail can be explained by the predominance of this system at rest in this line. In conclusion, both the basal autonomic activity and the autonomic responses differed according to the emotional reactivity, and changes in autonomic activity appear to be related to the genetic selection process.

Elevated anxiety-like and depressive behavior in Desert hedgehog knockout male mice.

To investigate the functional role of Desert hedgehog (Dhh) gene in the nervous system, we examined motor, sensory, learning and memory functions as well as mood in Dhh knockout (KO) mice. Dhh KO male mice exhibited prolonged immobility time compared with wild-type male mice in the forced swimming test, and showed enhanced inhibition in the Vogel's conflict model. These findings suggest that Dhh KO male mice exhibited enhanced anxiety and depressive behavior compared with wild-type male mice. In contrast, Dhh KO female mice did not show any significant difference compared to wild-type female mice. These behavioral abnormalities of Dhh KO male mice may be due to lower testosterone levels with abnormal development of the testes caused by Dhh-null mutation.

Behavioural alterations in male mice lacking the gene for D-aspartate oxidase.

D-serine and D-aspartate are important regulators of mammalian physiology. D-aspartate is found in nervous and endocrine tissue, specifically in hypothalamic supraoptic and paraventricular nuclei, pituitary, and adrenal medullary cells. Endogenous D-aspartate is selectively degraded by D-aspartate oxidase. We previously reported that adult male mice lacking the gene for D-aspartate oxidase (Ddo(-/-) mice) display elevated concentrations of D-aspartate in several neuronal and neuroendocrine tissues as well as impaired sexual performance and altered autogrooming behaviour. In the present study, we analyzed behaviours relevant to affect, cognition, and motor control in Ddo(-/-) mice. Ddo(-/-) mice display deficits in sensorimotor gating and motor coordination as well as reduced immobility in the forced swim test. Basal corticosterone concentrations are elevated. The Ddo(-/-) mice have D-aspartate immunoreactive cells in the cerebellum and adrenal glands that are not observed in the wild-type mice. However, no differences in anxiety-like behaviour are detected in open field or light-dark preference tests. Also, Ddo(-/-) mice do not differ from wild-type mice in either passive avoidance or spontaneous alternation tasks. Although many of these behavioural deficits may be due to the lack of Ddo during development, our results are consistent with the widespread distribution of D-aspartate and the hypothesis that endogenous D-aspartate serves diverse behavioural functions.