Neonatal anoxia in rats: hippocampal cellular and subcellular changes related to cell death and spatial memory.

Neonatal anoxia in rodents has been used to understand brain changes and cognitive dysfunction following asphyxia. This study investigated the time-course of cellular and subcellular changes and hippocampal cell death in a non-invasive model of anoxia in neonatal rats, using Terminal deoxynucleotidyl transferase-mediated dUTP Nick End Labeling (TUNEL) to reveal DNA fragmentation, Fluoro-Jade® B (FJB) to show degenerating neurons, cleaved caspase-3 immunohistochemistry (IHC) to detect cells undergoing apoptosis, and transmission electron microscopy (TEM) to reveal fine ultrastructural changes related to cell death. Anoxia was induced by exposing postnatal day 1 (P1) pups to a flow of 100% gaseous nitrogen for 25 min in a chamber maintained at 37 °C. Control rats were similarly exposed to this chamber but with air flow instead of nitrogen. Brain changes following anoxia were evaluated at postnatal days 2, 14, 21 and 60 (P2, P14, P21 and P60). In addition, spatial reference memory following anoxia and control treatments was evaluated in the Morris water maze, starting at P60. Compared to their respective controls, P2 anoxic rats exhibited (1) higher TUNEL labeling in cornus ammonis (CA) 1 and the dentate gyrus (DG), (2) higher FJB-positive cells in the CA2-3, and (3) somato-dendritic swelling, mitochondrial injury and chromatin condensation in irregular bodies, as well as other subcellular features indicating apoptosis, necrosis, autophagy and excitotoxicity in the CA1, CA2-3 and DG, as revealed by TEM. At P14, P21 and P60, both groups showed small numbers of TUNEL-positive and FJB-positive cells. Stereological analysis at P2, P14, P21 and P60 revealed a lack of significant differences in cleaved caspase-3 IHC between anoxic and control subjects. These results suggest that the type of hippocampal cell death following neonatal anoxia is likely independent of caspase-3 activation. Neonatal anoxia induced deficits in acquisition and performance of spatial reference memory in the Morris water maze task. Compared to control subjects, anoxic animals exhibited increased latencies and path lengths to reach the platform, as well as decreased searching specifically for the platform location. In contrast, no significant differences were observed for swimming speeds and frequency within the target quadrant. Together, these behavioral results indicate that the poorer performance by anoxic subjects is related to spatial memory deficits and not to sensory or motor deficits. Therefore, this model of neonatal anoxia in rats induces hippocampal changes that result in cell losses and impaired hippocampal function, and these changes are likely related to spatial memory deficits in adulthood.

Long-term social recognition memory in adult male rats: factor analysis of the social and non-social behaviors

A modified version of the intruder-resident paradigm was used to investigate if social recognition memory lasts at least 24 h. One hundred and forty-six adult male Wistar rats were used. Independent groups of rats were exposed to an intruder for 0.083, 0.5, 2, 24, or 168 h and tested 24 h after the first encounter with the familiar or a different conspecific. Factor analysis was employed to identify associations between behaviors and treatments. Resident rats exhibited a 24-h social recognition memory, as indicated by a 3- to 5-fold decrease in social behaviors in the second encounter with the same conspecific compared to those observed for a different conspecific, when the duration of the first encounter was 2 h or longer. It was possible to distinguish between two different categories of social behaviors and their expression depended on the duration of the first encounter. Sniffing the anogenital area (49.9 percent of the social behaviors), sniffing the body (17.9 percent), sniffing the head (3 percent), and following the conspecific (3.1 percent), exhibited mostly by resident rats, characterized social investigation and revealed long-term social recognition memory. However, dominance (23.8 percent) and mild aggression (2.3 percent), exhibited by both resident and intruders, characterized social agonistic behaviors and were not affected by memory. Differently, sniffing the environment (76.8 percent of the non-social behaviors) and rearing (14.3 percent), both exhibited mostly by adult intruder rats, characterized non-social behaviors. Together, these results show that social recognition memory in rats may last at least 24 h after a 2-h or longer exposure to the conspecific.

Long-term social recognition memory in adult male rats: factor analysis of the social and non-social behaviors.

A modified version of the intruder-resident paradigm was used to investigate if social recognition memory lasts at least 24 h. One hundred and forty-six adult male Wistar rats were used. Independent groups of rats were exposed to an intruder for 0.083, 0.5, 2, 24, or 168 h and tested 24 h after the first encounter with the familiar or a different conspecific. Factor analysis was employed to identify associations between behaviors and treatments. Resident rats exhibited a 24-h social recognition memory, as indicated by a 3- to 5-fold decrease in social behaviors in the second encounter with the same conspecific compared to those observed for a different conspecific, when the duration of the first encounter was 2 h or longer. It was possible to distinguish between two different categories of social behaviors and their expression depended on the duration of the first encounter. Sniffing the anogenital area (49.9% of the social behaviors), sniffing the body (17.9%), sniffing the head (3%), and following the conspecific (3.1%), exhibited mostly by resident rats, characterized social investigation and revealed long-term social recognition memory. However, dominance (23.8%) and mild aggression (2.3%), exhibited by both resident and intruders, characterized social agonistic behaviors and were not affected by memory. Differently, sniffing the environment (76.8% of the non-social behaviors) and rearing (14.3%), both exhibited mostly by adult intruder rats, characterized non-social behaviors. Together, these results show that social recognition memory in rats may last at least 24 h after a 2-h or longer exposure to the conspecific.

Financial support of graduate programs in Brazil: quo vadis?

Graduate programs provide the highest level of formal education and thus are crucial for the development of any country. However, official Brazilian data clearly show a dramatic decrease in the number and values of scholarships available to graduate programs in Brazil over the last few years, despite the importance and growth of such programs. Between 1995 and 2004, investment by the Coordenadoria de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES, subordinate to the Ministry of Education and Culture) in funding scholarships, corrected for inflation in the period, actually decreased by 51%. In addition, during the period between 1994 and 2004, there was a loss of about 60% in the purchasing power of the graduate scholarships provided by CAPES and the National Council for Science and Technology (CNPq). To reverse this trend, we propose the development of sectorial funding for Brazilian graduate programs to guarantee the availability and continuity of financial support for this strategic activity.

Financial support of graduate programs in Brazil: quo vadis?

Graduate programs provide the highest level of formal education and thus are crucial for the development of any country. However, official Brazilian data clearly show a dramatic decrease in the number and values of scholarships available to graduate programs in Brazil over the last few years, despite the importance and growth of such programs. Between 1995 and 2004, investment by the Coordenadoria de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES, subordinate to the Ministry of Education and Culture) in funding scholarships, corrected for inflation in the period, actually decreased by 51 percent. In addition, during the period between 1994 and 2004, there was a loss of about 60 percent in the purchasing power of the graduate scholarships provided by CAPES and the National Council for Science and Technology (CNPq). To reverse this trend, we propose the development of sectorial funding for Brazilian graduate programs to guarantee the availability and continuity of financial support for this strategic activity.

Working memory and acquisition of implicit knowledge by imagery training, without actual task performance.

This study investigated acquisition of a mirror-reading skill via imagery training, without the actual performance of a mirror-reading task. In experiment I, healthy volunteers simulated writing on an imaginary, transparent screen placed at eye level, which could be read by an experimenter facing the subject. Performance of this irrelevant motor task required the subject to imagine the letters inverted, as if seen in a mirror from their own point of view (imagery training). A second group performed the same imagery training interspersed with a complex, secondary spelling and counting task. A third, control, group simply wrote the words as they would normally appear from their own point of view. After training with 300 words, all subjects were tested in a mirror-reading task using 60 non-words, constructed according to acceptable letter combinations of the Portuguese language. Compared with control subjects, those exposed to imagery training, including those who switched between imagery and the complex task, exhibited shorter reading times in the mirror-reading task. Experiment II employed a 2 x 3 design, including two training conditions (imagery and actual mirror-reading) and three competing task conditions (a spelling and counting switching task, a visual working memory concurrent task, and no concurrent task). Training sessions were interspersed with mirror-reading testing sessions for non-words, allowing evaluation of the mirror-reading acquisition process during training. The subjects exposed to imagery training acquired the mirror-reading skill as quickly as those exposed to the actual mirror-reading task. Further, performance of concurrent tasks together with actual mirror-reading training severely disrupted mirror-reading skill acquisition; this interference effect was not seen in subjects exposed to imagery training and performance of the switching and the concurrent tasks. These results unequivocally show that acquisition of implicit skills by top-down imagery training is at least as efficient as bottom-up acquisition.

Dentate gyrus-selective colchicine lesion and disruption of performance in spatial tasks: difficulties in "place strategy" because of a lack of flexibility in the use of environmental cues?

The effects of intradentate colchicine injections on the performance of tasks requiring spatial working and reference memory are controversial. Multiple-site colchicine injections (7 microg/microl; via a drawn micropipette) throughout the dentate gyrus (DG) of rats (nine sites in each hemisphere, 0.06 microl at each site) selectively destroy about 90% of the DG granule cells, as revealed by quantitative stereological estimates; stereology also revealed minor neuronal losses in the CA4 (33%) and CA1 (23%) subfields, but lack of damage to the CA3 hippocampal subfield. Spatial reference and working memory were assessed in Morris' water maze; in the reference memory task, the rats were required to learn a single, fixed location for the platform over several days of training; in the working memory task, animals were required to learn a new platform location every day, in a matching-to-place procedure. Compared to sham-operated controls, lesioned rats showed significant disruption in acquisition of the reference memory water maze task; however, the data reveal that these rats did acquire relevant information about the task, probably based on guidance and orientation strategies. In a subsequent probe test, with the platform removed, lesioned rats showed disruption in precise indexes of spatial memory (e.g., driving search towards the surroundings of the former platform location), but not in less precise indexes of spatial location. Finally, the lesioned rats showed no improvement in the match-to-place procedure, suggesting that their working memory for places was disrupted. Thus, although capable of acquiring relevant information about the task, possibly through guidance and/or orientation strategies, DG-lesioned rats exhibit a marked difficulty in place strategies. This is particularly evident when these rats are required to deal with one-trial place learning in a familiar environment, such as in the working memory version of the water maze task, which requires flexibility in the use of previously acquired information.

Loss of CA1 cells following global ischaemia correlates with spatial deficits in the circular platform task.

The effect of 15 min, four-vessel-occlusion (4-VO) ischaemia on performance by rats in the circular platform task (CPT) was investigated. Possible correlations between the extent of hippocampal cell loss and behavioural disruption were evaluated. Sham-operated controls (n=10) and 4-VO ischaemic animals (n=32) were required to escape from a 1.2 m diameter, brightly illuminated, white surface into a dark goal box located under one of 18 equally-spaced, 9 cm diameter holes arranged around the circumference (3 trials per day). The goal box was maintained in a single, fixed, rewarded location relative to the extramaze cues for 7 days (days 16-22 post-ischaemia). During the reversal test, the goal box was transferred to a new location 140 degrees from the initial point and kept in this new position from day 23 through day 25 post-ischaemia. Ischaemic rats were slower to find the goal box than sham-operated controls; this learning deficit correlated with the degree of neuronal loss in the CA1, but not in the CA2, CA3 and CA4 subfields and presubiculum of the hippocampal formation. During the reversal test, ischaemic rats persisted in searching for the goal box at the initially rewarded location. The circular platform task provides a good model for behavioural studies following transient forebrain ischaemia in the rat.

Hippocampal lesions induced by ionizing radiation: a parametric study.

The selective lesion of granule cell populations in the dentate gyrus induced by ionizing radiation has been proposed as a useful method for evaluating the effects of hippocampal lesions on behavioral tasks. In the first part of the present study we confirmed the induction of the selective lesion of hippocampal dentate gyrus by ionizing radiation in infant Wistar rats, reported previously, but to a smaller extent with less cell loss. A parametric study was thus performed to assess the effect of modification of the parameters previously tested, comprising three further steps: an increase in the total dose of X-rays and modification of the fractionating schedule; use of three radiation types, X-ray, gamma-ray, and electrons (at two energy levels, 3 and 7 mev); use of three X-ray energy levels, 180, 200 and 250 kVp; and assessment of the effect of five total X-ray doses, at 200 kVp, 10, 14, 16, 18 and 20 gy (grays). The data suggests that X-ray radiation, in a total dose of 14 gy, at the 200 kVp energy level, fractionated into seven consecutive exposures of 2 gy each and produces a lesion of about 85% of the dentate gyrus granule cells.

Rats do react to stimulus omission.

1. The great majority of data supporting the hypothesis of a system capable of comparing current sensorial inputs with an internal representation of the environment comes from studies about exploratory activity to new stimuli or to manipulation of features of a familiar stimulus. On the other hand, these data could also be explained simply by arousal constructs. In this context, demonstrations of exploratory behavior to the absence of a previously presented stimulus (i.e., stimulus omission) would provide stronger support for the idea of a comparator. 2. To test the reaction of rats to the absence of a stimulus, rats were submitted to 7 exploratory trials in an open-field. In the 1st trial there were only two patterns on the apparatus wall. In trials 2-6 a stimulus was presented in a designated area of the field. Finally, in the 7th trial this stimulus was omitted. Results showed that the animals reacted to the stimulus omission by spending more time in the stimulus presentation place during the 7th trial than 1) in the 1st trial (also without stimulus), 2) in the 6th trial (last trial with a stimulus present), and 3) in 3 neutral sectors of the same size as the stimulus presentation place, during the 7th trial. 3. These data indicate that rats do react to the absence of a familiar stimulus and provide strong support for the existence of a Comparator System since the rats responded to "something that wasn't there anymore", a response that could only be due to a reaction triggered by a mismatch between internal representation of the environment and its present state.

Rats do react to stimulus omission

1. The great majority of data supporting the hypothesis of a systemcapable of compararing current sensorial imputs with and internal representation of the environment comes from studies about exploratory activity to new stimuli or to manipulation of features of a familiar stimulus. On the other hand, these data could also be explained simply by arousal constructs. In this context, demonstrations of exploratory behavior to the absence of a previously presented stimulus (i.e., stimulus omission) would provide stronger support for the idea of a comparator. 2. To test the reaction of rats to the absence of a stimulus, rats were submitted to 7 exploratory trials in an open-field. In the 1st trial there were only two patterns on the apparatus wall. In trials 2-6 a stimulus was presented in a designated area of the field. Finally, in the 7th trial this stimulus was omitted. Results showed that the animals reacted to the stimulus omission by spending more time in the stimulus presentation place during the 7th trial than 1) in the 1st trial (also without stimulus), 2) in the 6th trial (last trial with a stimulus present), and 3) in 3 neutral sectors of the same size as the stimulus presentation place, during the 7th trial. 3. These data indicated thath rats do react to the absence of a familiar stimulus and provide strong support for the existence of a Comparator System since the rats responded to "something that wasn't there anymore", a response that could only be due to a reaction triggered by a mismatch between internal representation of the environment and its present state

Induction of microglial immunomolecules by anterogradely degenerating mossy fibres in the rat hippocampal formation.

Degeneration of myelinated axonal connections is generally held to provide a strong stimulus for microglial expression of major histocompatibility complex (MHC) class II antigen. The present study demonstrates that strong microglial reactions also are induced by axonal and terminal degeneration of the unmyelinated hippocampal mossy fibres. After destruction of dentate granule cells by focal injections of colchicine (or transection of the mossy fibres) in adult rats, immunocytochemical analysis of the mossy fibre terminal fields in the dentate hilus and regio inferior of hippocampus proper (CA3) revealed profound changes in microglial cells with increased expression of the complement receptor type 3 and induction of MHC class I antigen, leukocyte common antigen, lymphocyte function-associated antigen-1 and MHC class II antigen. The microglial reaction, first detectable 4 days after the lesion, became maximal during the third postlesional week, and had almost vanished 6 weeks after the lesion. From recent studies we know that anterograde degeneration of myelinated Schaffer-collaterals from CA3 to regio superior of hippocampus proper and myelinated entorhinal perforant path fibres to fascia dentata is accompanied by microglial expression of MHC class I antigen, but not class II. Together with the present findings, this demonstrates that myelin debris is neither necessary nor sufficient to induce expression of microglial MHC class II antigen within the hippocampus.

Rats with dorsal hippocampal lesions do react to new stimuli but not to spatial changes of known stimuli.

The effect of visual distracting stimuli upon the straight alleyway performance of dorsal hippocampectomized Wistar rats was investigated. In comparison with control animals it was observed that dorsal hippocampectomized animals (1) ambulated more during the preexposure phase, (2) acquired at the same rate a running response for food (training phase), (3) reacted similarly to a new visual stimulus (black cards) presented in a sector of the alleyway, and (4) habituated to successive presentations of that stimulus in the same place. (5) However, dorsal hippocampectomized rats did not react, unlike the controls, to the presentation of the same stimulus in another place of the alleyway but (6) reacted to the visual pattern change of the stimulus (now black/white check cards) in the same place. These results indicate that under certain experimental conditions, hippocampus-lesioned animals are capable of interrupting a running response for food in order to explore a new conspicuously located stimulus, habituate to repeated presentations of that stimulus, and to react to a new pattern of visual stimulation. They suggest that hippocampectomized rats do not lose the capacity to react to a new stimulus; the disruption seems to be related to the spatial context of stimulus presentation, supporting a spatial mapping hypothesis of hippocampal function.

On delay-of-punishment and preexposure time: effects on passive avoidance behavior in rats.

Some procedures commonly employed in testing hypotheses of instrumental learning and classical conditioning as applied to the step-through passive avoidance task in rats are examined. In Experiment I, a 'delay-of-punishment gradient', i.e., decreased passive avoidance performance, was obtained by increasing response-shock time intervals (from 0 to 600 s) in training sessions. In Experiment II, decreased passive avoidance behavior was also obtained by submitting animals to nonreinforced preexposure (0 to 600 s) in the shock compartment prior to their receiving shock contingent on the response in training sessions. Experiment III repeated the 'delay-of-punishment gradient' and showed that adequate nonreinforced preexposure time before training decreases passive avoidance performance in animals trained through a noncontingent procedure. The results clearly show that environment training preexposure has a decreasing effect on passive avoidance performance. Increasing response-shock interval also has a decremental effect on this task. With large time intervals the latter effect can be explained as the result of preexposure to the training environment; the possibility of interaction between both processes was not discarded. For smaller time ranges (up to 30 s), the usual interpretation of 'delay-of-punishment gradient', namely decreased response-shock association, seems to be adequate to account for the results.

Effects of housing conditions on three behavioral tests in rats.

Groups of rats housed since weaning under two different kinds of housing conditions (wire and wooden cages) were compared at adulthood in the open field test, the step-through passive avoidance test, and for aggressiveness induced by REM-sleep deprivation and apomorphine administration. In the open field test wire-caged rats showed less rearing and grooming than wood-caged rats. This difference was accentuated by a single previous electrical shock (i.e., wire-caged rats exhibited less ambulation, rearing and grooming and more defecation than wood-caged rats, after shock) and was not altered by prior habituation to handling. In the passive avoidance test there were no significant differences between wire- and wood-caged rats. Wire-caged rats were more aggressive than wood-caged rats after REM-sleep deprivation and the administration of apomorphine. These results show that the control of previous housing conditions is an important variable to be considered in behavioral studies.