Effect of repeated restraint stress on memory in different tasks

The present study investigated the effect of repeated stress applied to female rats on memory evaluated by three behavioral tasks: two-way shuttle avoidance, inhibitory avoidance and habituation to an open field. Repeated stress had different effects on rat behavior when different tasks were considered. In the two-way active avoidance test the stressed animals presented memory of the task, but their memory scores were impaired when compared to all other groups. In the habituation to the open field, only the control group showed a significant difference in the number of rearings between training and testing sessions, which is interpreted as an adequate memory of the task. In the handled and chronically stressed animals, on the other hand, no memory was observed, suggesting that even a very mild repeated stress would be enough to alter habituation to this task. The performance in the inhibitory avoidance task presented no significant differences between groups. The findings suggest that repeated restraint stress might induce cognitive impairments that are dependent on the task and on stress intensity.

Interaction between repeated restraint stress and concomitant midazolam administration on sweet food ingestion in rats

Emotional changes can influence feeding behavior. Previous studies have shown that chronically stressed animals present increased ingestion of sweet food, an effect reversed by a single dose of diazepam administered before testing the animals. The aim of the present study was to evaluate the response of animals chronically treated with midazolam and/or submitted to repeated restraint stress upon the ingestion of sweet food. Male adult Wistar rats were divided into two groups: controls and exposed to restraint 1 h/day, 5 days/week for 40 days. Both groups were subdivided into two other groups treated or not with midazolam (0.06 mg/ml in their drinking water during the 40-day treatment). The animals were placed in a lighted area in the presence of 10 pellets of sweet food (Froot loops®). The number of ingested pellets was measured during a period of 3 min, in the presence or absence of fasting. The group chronically treated with midazolam alone presented increased ingestion when compared to control animals (control group: 2.0 ± 0.44 pellets and midazolam group: 3.60 ± 0.57 pellets). The group submitted to restraint stress presented an increased ingestion compared to controls (control group: 2.0 ± 0.44 pellets and stressed group: 4.18 ± 0.58 pellets). Chronically administered midazolam reduced the ingestion in stressed animals (stressed/water group: 4.18 ± 0.58 pellets; stressed/midazolam group: 3.2 ± 0.49 pellets). Thus, repeated stress increases appetite for sweet food independently of hunger and chronic administration of midazolam can decrease this behavioral effect.

Effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices

It has been suggested that glucocorticoids released during stress might impair neuronal function by decreasing glucose uptake by hippocampal neurons. Previous work has demonstrated that glucose uptake is reduced in hippocampal and cerebral cortex slices 24 h after exposure to acute stress, while no effect was observed after repeated stress. Here, we report the effect of acute and repeated restraint stress on glucose oxidation to CO2 in hippocampal and cerebral cortex slices and on plasma glucose and corticosterone levels. Male adult Wistar rats were exposed to restraint 1 h/day for 50 days in the chronic model. In the acute model there was a single exposure. Immediately or 24 h after stress, the animals were sacrificed and the hippocampus and cerebral cortex were dissected, sliced, and incubated with Krebs buffer, pH 7.4, containing 5 mM glucose and 0.2 µCi D-[U-14C] glucose. CO2 production from glucose was estimated. Trunk blood was also collected, and both corticosterone and glucose were measured. The results showed that corticosterone levels after exposure to acute restraint were increased, but the increase was smaller when the animals were submitted to repeated stress. Blood glucose levels increased after both acute and repeated stress. However, glucose utilization, measured as CO2 production in hippocampal and cerebral cortex slices, was the same in stressed and control groups under conditions of both acute and chronic stress. We conclude that, although stress may induce a decrease in glucose uptake, this effect is not sufficient to affect the energy metabolism of these cells.

Effect of repeated stress on novelty-induced antinociception in rats

There is extensive evidence that acute stress induces an analgesic response in rats. On the other hand, repeatedly stressed animals may present the opposite effect, i.e., hyperalgesia. Furthermore, exposure to novelty is known to induce antinociception. The effects of repeated restraint stress on nociception after exposure to novelty, as measured by the tail-flick latency (TFL), were studied in adult male rats. The animals were stressed by restraint 1 h daily, 5 days a week for 40 days. The control group was not submitted to restraint. Nociception was assessed with a tail-flick apparatus. After being familiarized with the TFL apparatus, each group was subdivided into two other groups, i.e., with or without novelty. Animals were subjected to the TFL measurement twice. For the animals exposed to novelty, the first TFL measurement was made immediately before, and the second 2 min after a 2-min exposure to a new environment. While the control group presented an increased TFL after exposure to a novel environment, chronically stressed animals did not show this effect. These results suggest that repeated restraint stress induces an alteration in the nociceptive response, perhaps as a result of an alteration in endogenous opioids in these animals.

c-Fos expression induced by electroacupuncture at the Zusanli point in rats submitted to repeated immobilization

In laboratory animals, acupuncture needs to be performed on either anesthetized or, if unanesthetized, restrained subjects. Both procedures up-regulate c-Fos expression in several areas of the central nervous system, representing therefore a major pitfall for the assessment of c-Fos expression induced by electroacupuncture. Thus, in order to reduce the effect of acute restraint we used a protocol of repeated restraint for the assessment of the brain areas activated by electroacupuncture in adult male Wistar rats weighing 180-230 g. Repeated immobilization protocols (6 days, 1 h/day and 13 days, 2 h/day) were used to reduce the effect of acute immobilization stress on the c-Fos expression induced by electroacupuncture at the Zusanli point (EA36S). Animals submitted to immobilization alone or to electroacupuncture (100 Hz, 2-4 V, faradic wave) in a non-point region were compared to animals submitted to electroacupuncture at EA36S (4 animals/subgroup). c-Fos expression was measured in 41 brain areas by simple counting of cells and the results are reported as number of c-Fos-immunoreactive cells/10,000 µm². The protocols of repeated immobilization significantly reduced the immobilization-induced c-Fos expression in most of the brain areas analyzed (P < 0.05). Animals of the EA36S groups had significantly higher levels of c-Fos expression in the dorsal raphe nucleus, locus coeruleus, posterior hypothalamus and central medial nucleus of the thalamus. Furthermore, the repeated immobilization protocols intensified the differences between the effects of 36S and non-point stimulation in the dorsal raphe nucleus (P < 0.05). These data suggest that high levels of stress can interact with and mask the evaluation of specific effects of acupuncture in unanesthetized animals.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Changes in the biogenic amine content of the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens of rats submitted to single and repeated sessions of the elevated plus-maze test

It has been demonstrated that exposure to a variety of stressful experiences enhances fearful reactions when behavior is tested in current animal models of anxiety. Until now, no study has examined the neurochemical changes during the test and retest sessions of rats submitted to the elevated plus maze (EPM). The present study uses a new approach (HPLC) by looking at the changes in dopamine and serotonin levels in the prefrontal cortex, amygdala, dorsal hippocampus, and nucleus accumbens in animals upon single or double exposure to the EPM (one-trial tolerance). The study involved two experiments: i) saline or midazolam (0.5 mg/kg) before the first trial, and ii) saline or midazolam before the second trial. For the biochemical analysis a control group injected with saline and not tested in the EPM was included. Stressful stimuli in the EPM were able to elicit one-trial tolerance to midazolam on re-exposure (61.01%). Significant decreases in serotonin contents occurred in the prefrontal cortex (38.74%), amygdala (78.96%), dorsal hippocampus (70.33%), and nucleus accumbens (73.58%) of the animals tested in the EPM (P < 0.05 in all cases in relation to controls not exposed to the EPM). A significant decrease in dopamine content was also observed in the amygdala (54.74%, P < 0.05). These changes were maintained across trials. There was no change in the turnover rates of these monoamines. We suggest that exposure to the EPM causes reduced monoaminergic neurotransmission activity in limbic structures, which appears to underlie the "one-trial tolerance" phenomenon.

Automatic recognition of fish from video sequences

The problem of automatic recognition of the fish from the video sequences is discussed in this Master’s Thesis. This is a very urgent issue for many organizations engaged in fish farming in Finland and Russia because the process of automation control and counting of individual species is turning point in the industry. The difficulties and the specific features of the problem have been identified in order to find a solution and propose some recommendations for the components of the automated fish recognition system. Methods such as background subtraction, Kalman filtering and Viola-Jones method were implemented during this work for detection, tracking and estimation of fish parameters. Both the results of the experiments and the choice of the appropriate methods strongly depend on the quality and the type of a video which is used as an input data. Practical experiments have demonstrated that not all methods can produce good results for real data, whereas on synthetic data they operate satisfactorily.

Repeated exposure of adolescent rats to oral methylphenidate does not induce behavioral sensitization or cross-sensitization to nicotine

Several lines of evidence indicate that the use of stimulant drugs, including methylphenidate (MPD), increases tobacco smoking. This has raised concerns that MPD use during adolescence could facilitate nicotine abuse. Preclinical studies have shown that repeated treatment with an addictive drug produces sensitization to that drug and usually cross-sensitization to other drugs. Behavioral sensitization has been implicated in the development of drug addiction. We examined whether repeated oral MPD administration during adolescence could induce behavioral sensitization to MPD and long-lasting cross-sensitization to nicotine. Adolescent male Wistar rats were treated orally with 10 mg/kg MPD or saline (SAL) from postnatal day (PND) 27 to 33. To evaluate behavioral sensitization to MPD in adolescent rats (PND 39), the SAL pretreated group was subdivided into two groups that received intragastric SAL (1.0 mL/kg) or MPD (10 mg/kg); MPD pretreated rats received MPD (10 mg/kg). Cross-sensitization was evaluated on PND 39 or PND 70 (adulthood). To this end, SAL- and MPD-pretreated groups received subcutaneous injections of SAL (1.0 mL/kg) or nicotine (0.4 mg/kg). All groups had 8 animals. Immediately after injections, locomotor activity was determined. The locomotor response to MPD challenge of MPD-pretreated rats was not significantly different from that of the SAL-pretreated group. Moreover, the locomotor response of MPD-pretreated rats to nicotine challenge was not significantly different from that of the SAL-pretreated group. This lack of sensitization and cross-sensitization suggests that MPD treatment during adolescence does not induce short- or long-term neuroadaptation in rats that could increase sensitivity to MPD or nicotine.

Stability of relative oxygen pulse curve during repeated maximal cardiopulmonary testing in professional soccer players

During cardiopulmonary exercise testing (CPET), stroke volume can be indirectly assessed by O2 pulse profile. However, for a valid interpretation, the stability of this variable over time should be known. The objective was to analyze the stability of the O2 pulse curve relative to body mass in elite athletes. VO2, heart rate (HR), and relative O2 pulse were compared at every 10% of the running time in two maximal CPETs, from 2005 to 2010, of 49 soccer players. Maximal values of VO2 (63.4 ± 0.9 vs 63.5 ± 0.9 mL O2•kg-1•min-1), HR (190 ± 1 vs188 ± 1 bpm) and relative O2 pulse (32.9 ± 0.6 vs 32.6 ± 0.6 mL O2•beat-1•kg-1) were similar for the two CPETs (P > 0.05), while the final treadmill velocity increased from 18.5 ± 0.9 to 18.9 ± 1.0 km/h (P < 0.01). Relative O2 pulse increased linearly and similarly in both evaluations (r² = 0.64 and 0.63) up to 90% of the running time. Between 90 and 100% of the running time, the values were less stable, with up to 50% of the players showing a tendency to a plateau in the relative O2 pulse. In young healthy men in good to excellent aerobic condition, the morphology of the relative O2 pulse curve is consistent up to close to the peak effort for a CPET repeated within a 1-year period. No increase in relative O2pulse at peak effort could represent a physiologic stroke volume limitation in these athletes.

Anxiolytic effects of repeated treatment with an essential oil from Lippia alba and (R)-(-)-carvone in the elevated T-maze

Lippia alba (Mill.) N.E. Brown (Verbenaceae) is widely used in different regions of Central and South America as a tranquilizer. The plant’s anxiolytic properties, however, merit investigation. The present study evaluated the effects of repeated daily (14 days) intraperitoneal (ip) treatment with an essential oil (EO) from a chemotype of L. alba (LA, chemotype II, 12.5 and 25 mg/kg; N = 6-8) and (R)-(-)-carvone (25 mg/kg; N = 8-12), the main constituent of this chemotype, on male Wistar rats (weighing 250 g at the beginning of the experiments) submitted to the elevated T-maze (ETM). The ETM allows the measurement of two defensive responses: inhibitory avoidance and one-way escape. In terms of psychopathology, these responses have been related to generalized anxiety and panic disorder, respectively. Treatment with the EO impaired ETM avoidance latencies, without altering escape, in a way similar to the reference drug diazepam (P < 0.05) (avoidance 2: control = 84.6 ± 35.2; EO 12.5 mg/kg = 11.8 ± 3.8; EO 25 mg/kg = 14.6 ± 2.7; diazepam = 7 ± 2.1). (R)-(-)-carvone also significantly altered this same response (P < 0.05; avoidance 1: control = 91.9 ± 31.5; carvone = 11.6 ± 1.8; diazepam = 8.1 ± 3.3). These results were not due to motor changes since no significant effects were detected in an open field. These observations suggest that LA exerts anxiolytic-like effects on a specific subset of defensive behaviors that have been implicated in generalized anxiety disorder, and suggest that carvone is one of the constituents of LA responsible for its action as a tranquilizer.

Metal-Ion-Binding Oligonucleotides: High-Affinity Probes for Nucleic Acid Sequences

Small non-coding RNAs have numerous biological functions in cell and are divided into different classes such as: microRNA, snoRNA, snRNA and siRNA. MicroRNA (miRNA) is the most studied non-coding RNA to date and is found in plants, animals and some viruses. miRNA with short sequences is involved in suppressing translation of target genes by binding to their mRNA post-transcriptionally and silencing it. Their function besides silencing of the viral gene, can be oncogenic and therefore the cause of cancer. Hence, their roles are highlighted in human diseases, which increases the interest in using them as biomarkers and drug targets. One of the major problems to overcome is recognition of miRNA. Owing to a stable hairpin structure, chain invasion by conventional Watson-Crick base-pairing is difficult. One way to enhance the hybridization is exploitation of metal-ion mediated base-pairing, i. e. oligonucleotide probes that tightly bind a metal ions and are able to form a coordinative bonds between modified and natural nucleobases. This kind of metallo basepairs containing short modified oligonucleotides can also be useful for recognition of other RNA sequences containing hairpin-like structural motives, such as the TAR sequence of HIV. In addition, metal-ion-binding oligonucleotides will undoubtedly find applications in DNA-based nanotechnology. In this study, the 3,5-dimethylpyrazol-1-yl substituted purine derivatives were successfully incorporated within oligonucleotides, into either a terminal or non-terminal position. Among all of the modified oligonucleotides studied, a 2-(3,5-dimethylpyrazol-1-yl)-6-oxopurine base containing oligonucleotide was observed to bind most efficiently to their unmodified complementary sequences in the presence of both Cu2+ or Zn2+. The oligonucleotide incorporating 2,6-bis(3,5-dimethylpyrazol-1-yl)purine base also markedly increased the stability of duplexes in the presence of Cu2+ without losing the selectivity.

Construction of bovine adenovirus type 3 E1 and E3, substitution plasmids and the sequencing analysis of DNA pol and pTP /

The relative ease to concentrate and purify adenoviruses, their well characterized mid-sized genome, and the ability to delete non-essential regions from their genome to accommodate foreign gene, made adenoviruses a suitable candidate for the construction of vectors. The use of adenoviral vectors in gene therapy, vaccination, and as a general vector system for expressing foreign genes have been documented for some time. In this study, the objective was to rescue a BAV3 E1 or E3 recombinant vector carrying the kanamycin resistant gene, a dominant selectable marker with useful applications in studying vectored gene expression in mammalian cells. To accomplish the objective of this study, more information about BAV3 DNA sequences was required in order to make the manipulation of the virus genome accessible. Therefore, sequencing of the BAV3 genome from 1 1 .7% to 30.8% was carried out. Analysis of the determined sequences revealed the primary structure of important viral gene products coded by E2 including BAV3 DNA pol and precursor to terminal protein. Comparative analysis of these proteins with their counterparts from human and non human adenoviruses revealed important insights as to the evolutionary lineage of BAV3. In order to insert the kanamycin resistance gene in either E1 or E3, it was necessary to delete BAV3 sequences to accommodate the foreign gene so as not to exceed the limit of the packaging capacity of the virus. To construct a recombinant BAV3 in which a foreign gene was inserted in the deleted E1 region, an E1 shuttle vector was constructed. This involved the deletion from the viral sequences a region between 1.3% to 9% and inserting the kanamycin resistance gene to replace the deletion. The E1 shuttle vector contained the left (0%- 53.9%) segment of the genome and was expected to generate BAV3 recombinants that can be grown and propagated in cells that can complement the missing E1 functions. To construct a similar shuttle vector for E3 deletion, DNA sequences extending from 78.9% to 82.5% (1281 bp) were deleted from within the E3 region that had been cloned into a plasmid vector. The deleted region corresponds to those that have been shown to be non-essential for viral replication in cell culture. The resulting plasmid was used to construct another recombinant plasmid with BAV3 DNA sequences extending from 37.1% to 100% and with a deletion of E3 sequences that were replaced by kanamycin resistance gene. This shuttle plasmid was used in cotransfections with digested viral DNA in an attempt to rescue a recombinant BAV3 carrying the kanamycin resistance gene to replace the deleted E3. In spite of repeated attempts of transfection, El or E3 recombinant BAV3 were not isolated. It seems that other approaches should be applied to make a final conclusion on BAV3 infectivity.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

A comparison of two computer-assisted remedial reading programs for adolescent unskilled readers : component reading skills and repeated reading

This study compared the relative effectiveness of two computerized remedial reading programs in improving the reading word recognition, rate, and comprehension of adolescent readers demonstrating significant and longstanding reading difficulties. One of the programs involved was Autoskill Component Reading Subskills Program, which provides instruction in isolated letters, syllables, and words, to a point of rapid automatic responding. This program also incorporates reading disability subtypes in its approach. The second program, Read It Again. Sam, delivers a repeated reading strategy. The study also examined the feasibility of using peer tutors in association with these two programs. Grade 9 students at a secondary vocational school who satisfied specific criteria with respect to cognitive and reading ability participated. Eighteen students were randomly assigned to three matched groups, based on prior screening on a battery of reading achievement tests. Two I I groups received training with one of the computer programs; the third group acted as a control and received the remedial reading program offered within the regular classroom. The groups met daily with a trained tutor for approximately 35 minutes, and were required to accumulate twenty hours of instruction. At the conclusion of the program, the pretest battery was repeated. No significant differences were found in the treatment effects of the two computer groups. Each of the two treatment groups was able to effect significantly improved reading word recognition and rate, relative to the control group. Comprehension gains were modest. The treatment groups demonstrated a significant gain, relative to the control group, on one of the three comprehension measures; only trends toward a gain were noted on the remaining two measures. The tutoring partnership appeared to be a viable alternative for the teacher seeking to provide individualized computerized remedial programs for adolescent unskilled readers. Both programs took advantage of computer technology in providing individualized drill and practice, instant feedback, and ongoing recordkeeping. With limited cautions, each of these programs was considered effective and practical for use with adolescent unskilled readers.