Contextual Fear Conditioning in Maternal Separated Rats: The Amygdala as a Site for Alterations

The first 2 weeks of life are a critical period for neural development in rats. Repeated long-term separation from the dam is considered to be one of the most potent stressors to which rat pups can be exposed, and permanently modifies neurobiological and behavioral parameters. Prolonged periods of maternal separation (MS) usually increase stress reactivity during adulthood, and enhance anxiety-like behavior. The aim of this study was to verify the effects of maternal separation during the neonatal period on memory as well as on biochemical parameters (Na⁺, K⁺-ATPase and antioxidant enzymes activities) in the amygdala of adult rats. Females and male Wistar rats were subjected to repeated maternal separation (incubator at 32 °C, 3 h/day) during postnatal days 1–10. At 60 days of age, the subjects were exposed to a Contextual fear conditioning task. One week after the behavioral task, animals were sacrificed and the amygdala was dissected for evaluation of Na⁺, K⁺-ATPase and antioxidant enzymes activities. Student-t test showed significant MS effect, causing an increase of freezing time in the three exposures to the aversive context in both sexes. Considering biochemical parameters Student-t test showed significant MS effect causing an increase of Na⁺, K⁺-ATPase activity in both sexes. On the other hand, no differences were found among the groups on the antioxidant enzymes activities [superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT)] in male rats, but in females, we found a significant MS effect, causing an increase of CAT activity and no differences were found among the groups on SOD and GPx activities. Our results suggest a role of early rearing environment in programming fear learning and memory in adulthood. An early stress experience such as maternal separation may increase activity in the amygdala (as pointed by the increased activity of Na⁺, K⁺-ATPase), affecting behaviors related to fear in adulthood, and this effect could be task-specific.     

Apelin-13 Impaires Acquisition but Not Consolidation or Expression of Contextual Fear in Rats

Apelin-13, as an endogenous neuropeptide, is the ligand for the G-protein-coupled receptor, APJ, which has recently been demonstrated to be involved in the process that contributes to learning and memory. Previous studies showed that apelin may be required for certain forms of learning and memory. Up to date, the role of apelin in fear memory has not been explored. In the present study, we tested the effects of apelin-13 (1.0, 2.0 and 4.0 µg/rat) on contextual fear conditioning (experiment 1), consolidation (experiment 2) and expression (experiment 3) in rats. A well established fear conditioning protocol was used, which contained three training phases: habituation, fear conditioning and test. Apelin-13 was i.c.v injected 10 min before conditioning (experiment 1), immediately after conditioning (experiment 2) or 10 min before testing (experiment 3). The values of percent freezing were used to measure fear. We found that only 2.0 µg apelin-13 administrations produced a decrease freezing in experiment 1. The most effective dose of apelin-13 (2.0 µg) was selected, but it had no effect on freezing in experiment 2 and 3. Furthermore, the decreased freezing in experiment 1 was not attributed to the deficits of locomotor activity and foot-shock sensitivity. These results, for the first time, indicated that apelin-13 impaired fear acquisition but not fear consolidation or expression.     

Ocean Acidification Disrupts Prey Responses to Predator Cues but Not Net Prey Shell Growth in Concholepas concholepas (loco)

Background

Most research on Ocean Acidification (OA) has largely focused on the process of calcification and the physiological trade-offs employed by calcifying organisms to support the building of calcium carbonate structures. However, there is growing evidence that OA can also impact upon other key biological processes such as survival, growth and behaviour. On wave-swept rocky shores the ability of gastropods to self-right after dislodgement, and rapidly return to normal orientation, reduces the risk of predation.

Methodology/Principal Findings

The impacts of OA on this self-righting behaviour and other important parameters such as growth, survival, shell dissolution and shell deposition in Concholepas concholepas (loco) were investigated under contrasting pCO₂ levels. Although no impacts of OA on either growth or net shell calcification were found, the results did show that OA can significantly affect self-righting behaviour during the early ontogeny of this species with significantly faster righting times recorded for individuals of C. concholepas reared under increased average pCO₂ concentrations (± SE) (716±12 and 1036±14 µatm CO₂) compared to those reared at concentrations equivalent to those presently found in the surface ocean (388±8 µatm CO₂). When loco were also exposed to the predatory crab Acanthocyclus hassleri, righting times were again increased by exposure to elevated CO₂, although self-righting times were generally twice as fast as those observed in the absence of the crab.

Conclusions and Significance

These results suggest that self-righting in the early ontogeny of C. concholepas will be positively affected by pCO₂ levels expected by the end of the 21st century and beginning of the next one. However, as the rate of self-righting is an adaptive trait evolved to reduce lethal predatory attacks, our result also suggest that OA may disrupt prey responses to predators in nature.     

Flavor Preference Learning Increases Olfactory and Gustatory Convergence onto Single Neurons in the Basolateral Amygdala but Not in the Insular Cortex in Rats

The basolateral amygdala (BLA) and the insular cortex (IC) represent two major areas for odor-taste associations, i.e. flavor integration. This learning may require the development of convergent odor and taste neuronal activation allowing the memory representation of such association. Yet identification of neurons that respond to such coincident input and the effect of flavor experience on odor-taste convergence remain unclear. In the present study we used the compartmental analysis of temporal activity using fluorescence in situ hybridization for Arc (catFISH) to visualize odor-taste convergence onto single neurons in the BLA and in the IC to assess the number of cells that were co-activated by both stimuli after odor-taste association. We used a sucrose conditioned odor preference as a flavor experience in rats, in which 9 odor-sucrose pairings induce a reliable odor-taste association. The results show that flavor experience induced a four-fold increase in the percentage of cells activated by both taste and odor stimulations in the BLA, but not in the IC. Because conditioned odor preference did not modify the number of cells responding selectively to one stimulus, this greater odor-taste convergence into individual BLA neurons suggests the recruitment of a neuronal population that can be activated by both odor and taste only after the association. We conclude that the development of convergent activation in amygdala neurons after odor-taste associative learning may provide a cellular basis of flavor memory.     

Repeated Idazoxan Increases Brain Imidazoline Receptors in Normotensive (WKY) but Not in Hypertensive (SHR) Rats

The specific binding of [3H]idazoxan in the presence of 10(-6) M (-)-adrenaline was used to evaluate the density of imidazoline receptors in the brain of spontaneously hypertensive (SHR) rats and sex- and age-matched normotensive Wistar-Kyoto (WKY) rats. In SHR rats the density of imidazoline receptors (cerebral cortex, hypothalamus, and medulla oblongata) was not different from that in normotensive (WKY) rats. However, repeated treatment with idazoxan consistently increased (23-80%) the density of imidazoline receptors in the various brain regions of WKY rats but not in SHR rats. In normotensive Sprague-Dawley rats, repeated treatment with the imidazoline drugs idazoxan and cirazoline also increased (33-37%) the density of imidazoline receptors in the cerebral cortex. The lack of regulation by idazoxan of the density of imidazoline receptors in the brain of SHR rats might reflect the existence of a relevant abnormality of these receptors in this genetic model of hypertension.     

Overexpression of Full-Length Centrobin Rescues Limb Malformation but Not Male Fertility of the Hypodactylous (hd) Rats

Rat hypodactyly (hd) mutation is characterized by abnormal spermatogenesis and sperm decapitation, limb malformation (missing digits II and III) and growth retardation. We have previously reported centrobin (centrosome BRCA2-interacting protein) truncation at the C-terminus in the hd mutant. Here, we report data from a transgenic rescue experiment carried out to determine a role of centrobin in pathogenesis of hd. The transgenic construct, consisting of full-length-coding cDNA linked to a ubiquitous strong promoter/enhancer combination, was inserted to chromosome 16 into a LINE repeat. No known gene is present in the vicinity of the insertion site. Transgenic centrobin was expressed in all tissues tested, including testis. Transgenic animals show normal body weight and limb morphology as well as average weight of testis and epididymis. Yet, abnormal spermatogenesis and sperm decapitation persisted in the transgenic animals. Western blotting showed the coexistence of full-length and truncated or partially degraded centrobin in sperm of the rescued transgenic animals. Immunocytochemistry showed a buildup of centrobin and ODF2 (outer dense fiber 2) at the sperm decapitation site in the hd mutant and rescued transgenic rats. Additional findings included bulge-like formations and thread-like focal dissociations along the sperm flagellum and the organization of multiple whorls of truncated sperm flagella in the epididymal lumen. We conclude that centrobin is essential for normal patterning of the limb autopod. Centrobin may be required for stabilizing the attachment of the sperm head to flagellum and for maintaining the structural integrity of the sperm flagellum. We postulate that the presence of truncated centrobin, coexisting with full-length centrobin, together with incorrect timing of transgenic centrobin expression may hamper the rescue of fertility in hd male rats.     

Basal Level of Autophagy Is Increased in Aging Human Skin Fibroblasts In Vitro,but Not in Old Skin

Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.     

Rhesus Monkeys (Macaca mulatta) Detect Rhythmic Groups in Music,but Not the Beat

It was recently shown that rhythmic entrainment, long considered a human-specific mechanism, can be demonstrated in a selected group of bird species, and, somewhat surprisingly, not in more closely related species such as nonhuman primates. This observation supports the vocal learning hypothesis that suggests rhythmic entrainment to be a by-product of the vocal learning mechanisms that are shared by several bird and mammal species, including humans, but that are only weakly developed, or missing entirely, in nonhuman primates. To test this hypothesis we measured auditory event-related potentials (ERPs) in two rhesus monkeys (Macaca mulatta), probing a well-documented component in humans, the mismatch negativity (MMN) to study rhythmic expectation. We demonstrate for the first time in rhesus monkeys that, in response to infrequent deviants in pitch that were presented in a continuous sound stream using an oddball paradigm, a comparable ERP component can be detected with negative deflections in early latencies (Experiment 1). Subsequently we tested whether rhesus monkeys can detect gaps (omissions at random positions in the sound stream; Experiment 2) and, using more complex stimuli, also the beat (omissions at the first position of a musical unit, i.e. the ‘downbeat’; Experiment 3). In contrast to what has been shown in human adults and newborns (using identical stimuli and experimental paradigm), the results suggest that rhesus monkeys are not able to detect the beat in music. These findings are in support of the hypothesis that beat induction (the cognitive mechanism that supports the perception of a regular pulse from a varying rhythm) is species-specific and absent in nonhuman primates. In addition, the findings support the auditory timing dissociation hypothesis, with rhesus monkeys being sensitive to rhythmic grouping (detecting the start of a rhythmic group), but not to the induced beat (detecting a regularity from a varying rhythm).     

Changes in Cholinergic but Not in GABAergic Markers in Amygdala, Piriform Cortex, and Nucleus Basalis of the Rat Brain Following Systemic Administration of Kainic Acid

Three days after systemic administration of kainic acid (15 mg/kg, s.c.), selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, and high-affinity choline uptake) and GABAergic parameters [benzodiazepine and gamma-aminobutyric acid (GABA) receptors] were studied in the frontal and piriform cortex, dorsal hippocampus, amygdaloid complex, and nucleus basalis. Kainic acid treatment resulted in a significant reduction of choline acetyltransferase activity in the piriform cortex (by 20%), amygdala (by 19%), and nucleus basalis (by 31%) in comparison with vehicle-injected control rats. A lower activity of acetylcholinesterase was also determined in the piriform cortex following parenteral kainic acid administration. [3H]Quinuclidinyl benzilate binding to muscarinic acetylcholine receptors was significantly decreased in the piriform cortex (by 33%), amygdala (by 39%), and nucleus basalis (by 33%) in the group treated with kainic acid, whereas such binding in the hippocampus and frontal cortex was not affected by kainic acid. Sodium-dependent high-affinity choline uptake into cholinergic nerve terminals was decreased in the piriform cortex (by 25%) and amygdala (by 24%) after kainic acid treatment. In contrast, [3H]flunitrazepam binding to benzodiazepine receptors and [3H]muscimol binding to GABA receptors were not affected 3 days after parenteral kainic acid application in any of the brain regions studied. The data indicate that kainic acid-induced limbic seizures result in a loss of cholinergic cells in the nucleus basalis that is paralleled by degeneration of cholinergic fibers and cholinoceptive structures in the piriform cortex and amygdala, a finding emphasizing the important role of cholinergic mechanisms in generating and/or maintaining seizure activity.     

Olfactory Conditioning of the Butterfly Agraulis vanillae (L.) (Lepidoptera, Nymphalidae) to Floral But Not Host-Plant Odors

The associative learning capacity of male and female nymphalid butterflies, Agraulis vanillae, was investigated. Both males and females were conditioned to chemical stimuli of amyl acetate and butyl acetate, but not of host-plant volatile emissions, although our EAG recordings demonstrate that Agraulis can detect host-plant aroma as well as both acetates. More female than male Agraulis were conditioned. Female butterflies reared in the laboratory generally exhibited a higher percentage of conditional responses than those collected in nature. The number of conditional responses on the first day of experiments was significantly smaller than on the ensuing 2–7 days.     

Inactivation of the Progesterone Receptor in Mx1+ Cells Potentiates Osteogenesis in Calvaria but Not in Long Bone

The effect of progesterone on bone remains elusive. We previously reported that global progesterone receptor (PR) knockout mice displayed high bone mass phenotype, suggesting that PR influences bone growth and modeling. Recently, Mx1+ cells were characterized to be mesenchymal stem cell-like pluripotent Cells. The aim of this study was to evaluate whether the PR in Mx1+ cells regulates osteogenesis. Using the Mx1-Cre;mT/mG reporter mouse model, we found that the calvarial cells exhibited minimal background Mx1-Cre activity prior to Cre activation by IFNα treatment as compared to the bone marrow stromal cells. IFNα treatment significantly activated Mx1-Cre in the calvarial cells. When the PR gene was deleted in the Mx1-Cre;PR-flox calvarial cells in vitro, significantly higher levels of expression of osteoblast maturation marker genes (RUNX2, Osteocalcin, and Dmp1) and osteogenic potential were detected. The PR-deficient calvariae exhibited greater bone volume, especially in the males. Although Mx1-Cre activity could be induced on the bone surface in vivo, the Mx1+ cells did not differentiate into osteocytes in long bones. Bone volumes at the distal femurs and the bone turnover marker serum Osteocalcin were similar between the Mx1-Cre;PR-flox mutant mice and the corresponding wild types in both sexes. In conclusion, our data demonstrates that blocking progesterone signaling via PRs in calvarial Mx1+ cells promoted osteoblast differentiation in the calvaria. Mx1+ was expressed by heterogeneous cells in bone marrow and did not differentiate into osteocyte during long bone development in vivo. Selectively inactivating the PR gene in Mx1+ cells affected the membrane bone formation but did not affect peripheral skeletal homeostasis.     

Long-Term Treatment of Clonidine,Atenolol, Amlodipine and Dihydrochlorothiazide,but Not Enalapril,Impairs the Sexual Function in Male Spontaneously Hypertensive Rats

This study was designed to investigate the impact of representative antihypertensive drugs of 5 classes on the sexual function in male spontaneously hypertensive rats (SHR) at doses that achieved similar blood pressure (BP) reduction. The experiment was performed in 6 groups of male SHR. The dose are 20 μg/kg/day for clonidine, 3 mg/kg/day for enalapril, 20 mg/kg/day for atenolol, 2 mg/kg/day for amlodipine, and 10 mg/kg/day for dihydrochlorothiazide. SHR were treated for 3 months, and then the penile erection and sexual behavior were detected. After BP recording, SHR were killed to evaluate the organ-damage, weight of accessory sex organs and levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH) and testosterone in serum. Five drugs had the similar efficacy on BP reduction. All drugs except of enalapril, significantly prolonged the mount latency, and decreased the mount frequency (P<0.05). Clonidine also reduced the conception rate (45% vs. 80% in control group, P<0.05). Amlodipine and dihydrochlorothiazide significantly increased the testosterone level (0.79±0.30, 0.80±0.34 vs. 0.49±0.20 in control group, unit: ng/dl, P<0.05). Enalapril, atenolol and amlodipine also significantly decreased the BP variability (systolic, 8.2±2.5, 7.6±1.8, 8.9±2.0 vs. 12.2±3.8 in control group, unit: mm Hg). All these drugs significantly decreased the organ-damage (P<0.05). In conclusion, long-term treatment with 5 common antihypertensive drugs possessed obvious organ protection in SHR. Clonidine, atenolol, amlodipine and dihydrochlorothiazide, but not enalapril, impair sexual function.     

Photoperiod Regulates Lean Mass Accretion,but Not Adiposity,in Growing F344 Rats Fed a High Fat Diet

In this study the effects of photoperiod and diet, and their interaction, were examined for their effects on growth and body composition in juvenile F344 rats over a 4-week period. On long (16L:8D), relative to short (8L:16D), photoperiod food intake and growth rate were increased, but percentage adiposity remained constant (ca 3-4%). On a high fat diet (HFD), containing 22.8% fat (45% energy as fat), food intake was reduced, but energy intake increased on both photoperiods. This led to a small increase in adiposity (up to 10%) without overt change in body weight. These changes were also reflected in plasma leptin and lipid levels. Importantly while both lean and adipose tissue were strongly regulated by photoperiod on a chow diet, this regulation was lost for adipose, but not lean tissue, on HFD. This implies that a primary effect of photoperiod is the regulation of growth and lean mass accretion. Consistent with this both hypothalamic GHRH gene expression and serum IGF-1 levels were photoperiod dependent. As for other animals and humans, there was evidence of central hyposomatotropism in response to obesity, as GHRH gene expression was suppressed by the HFD. Gene expression of hypothalamic AgRP and CRH, but not NPY nor POMC, accorded with the energy balance status on long and short photoperiod. However, there was a general dissociation between plasma leptin levels and expression of these hypothalamic energy balance genes. Similarly there was no interaction between the HFD and photoperiod at the level of the genes involved in thyroid hormone metabolism (Dio2, Dio3, TSHβ or NMU), which are important mediators of the photoperiodic response. These data suggest that photoperiod and HFD influence body weight and body composition through independent mechanisms but in each case the role of the hypothalamic energy balance genes is not predictable based on their known function.     

Decreased Hippocampal Expression, but Not Functionality, of GABAB Receptors After Transient Cerebral Ischemia in Rats

Abstract : We have examined the effects of transient global ischemia on both the gene expression levels and the functionality of GABA_B receptors in rat brain, using antisense in situ hybridization and electrophysiological evaluations. At the level of gene expression, no significant change in GABA_B receptor expression was observed in any hippocampal subfield at either 6 or 12 h after challenge. At 24 h postchallenge, however, a significant decrease in GABA_B receptor expression was observed in both the CA1 and CA3 subfields, whereas no change was observed in the dentate granule cell layer. Although expression in both the vulnerable CA1 and less vulnerable CA3 subfields was diminished at this time postchallenge, there was no significant difference in the degree of the diminished expression between these subfields. At the functional level, the dose-dependent ability of baclofen (1-100 μ M ) to inhibit an evoked excitatory postsynaptic potential (f-EPSP) in the CA1 subfield was evaluated at 24 h postischemia, in comparison with the dose-response observed in sham-operated subjects. No significant differences were observed in the efficacy of GABA_B receptor-mediated inhibition of the elicited f-EPSP at any of the baclofen concentrations examined. These data demonstrate that although the mRNA expression levels for the GABA_B receptor are diminished in both vulnerable and less vulnerable neurons of Ammon's horn at 24 h following transient global ischemia, the functionality of the GABA_B receptor system is maintained at this time postchallenge.     

Immune Investment Is Explained by Sexual Selection and Pace-of-Life,but Not Longevity in Parrots (Psittaciformes)

Investment in current reproduction should come at the expense of traits promoting future reproduction, such as immunity and longevity. To date, comparative studies of pace-of-life traits have provided some support for this, with slower paced species having greater immune function. Another means of investment in current reproduction is through secondary sexual characters (SSC). Investment in SSC''s is considered costly, both in terms of immunity and longevity, with greater costs being borne by species with more elaborate traits. Yet within species, females prefer more ornate males and those males are typically immunologically superior. Because of this, predictions about the relationship between immunity and SSC''s across species are not clear. If traits are costly, brighter species should have reduced immune function, but the opposite is true if SSC''s arise from selection for more immunocompetent individuals. My approach was to investigate immune investment in relation to SSC''s, pace-of-life and longevity while considering potentially confounding ecological factors. To do so I assessed leukocyte counts from in a novel group, the Psittaciformes. Investment in SSC''s best explained investment in immunity: species with brighter plumage had higher leukocyte counts and those with a greater degree of sexual dichromatism had fewer. Ecological variables and pace-of-life models tended to be poor predictors of immune investment. However, shorter incubation periods were associated with lower leukocyte counts supporting the notion that species with a fast pace-of-life invest less in immunity. These results suggest that investment in reproduction in terms of fast pace-of-life and sexual dichromatism results in reduced immunity; however, investment in plumage colour per se does not impose a cost on immunity across species.     

Phytotoxicity of Air Pollutants: Evidence for the Photodetoxification of SO(2) but Not O(3)

Pisum sativum L. cv Alsweet (garden pea) and Lycopersicon esculentum flacca Mill. (tomato) were used to evaluate the phytotoxicity of SO₂ and O₃ in the light and dark. Plants were grown in controlled environment chambers and exposed to SO₂ or O₃ in the light or dark at the same environmental conditions at which they were grown. The pea plants were treated with fusicoccin to ensure open stomata in the dark; the stomata of the tomato mutant remained open in the dark. Both species exhibited 64% to 80% less foliar necrosis following exposure to SO₂ (0.5 to 1.0 microliter per liter for 2 hours) in the light than in the dark. The decrease in SO₂ injury for light versus dark exposed plants was greater in fully expanded than expanding leaves. Both species exhibited 30% greater foliar necrosis following exposure to O₃ (0.2 microliter per liter for 2 hours) in the light than dark. The increase in O₃ injury in the light versus dark was similar for leaves at all stages of expansion. Leaf conductance to water vapor was 7% to 11% and 23% higher in the light than dark for fusicoccin-treated peas and tomato plants, respectively, indicating greater foliar uptake of both pollutants in the light than dark. Thus, the decreased SO₂ toxicity in the light was not associated with pollutant uptake, but rather the metabolism of SO₂. In contrast, the increased toxicity of O₃ in the light was at least in part associated with increased uptake or could not be separated from it.