Silvio Morato

Role of resocialization and of 5-HT1A receptor activation on the anxiogenic effects induced by isolation in the elevated plus-maze test

Rats were isolated for periods varying from 1 h to 2 weeks and the exploratory activity of these animals on the elevated plus-maze was studied. Rats isolated from periods of 2 h on displayed a significant reduction in the number of entries and time spent in the open arms of the plus-maze compared to socially housed controls. This effect was not correlated with the decrease in the total number of entries also produced by isolation. Acute treatment with midazolam or resocialization for a 24-h period clearly reversed these responses produced by prior 2-h isolation in the elevated plus-maze. It is suggested that exposure to a 2-h isolation period could be a useful nonpharmacological means of generating anxiety in laboratory rodents. Chronic treatment, but not acute treatment, with gepirone, a 5-HT1A agonist, inhibited the anxiogenic effects caused by a 2-week period of isolation. The reduction in aversiveness promoted by resocialization may be due to a recovery in the 5-HT activity depressed by isolation in a much faster way than observed with chronic gepirone administration.

Prenatal stress produces more behavioral alterations than maternal separation in the elevated plus-maze and in the elevated T-maze.

Prenatal stress and maternal separation are used in a large number of studies on early adversity consequences and present some similarities in their effects. The present work investigates the behavioral effects of these two procedures on two models of anxiety: the elevated plus-maze and the elevated T-maze. During pregnancy, female rats were submitted to uncontrollable electric foot shock sessions every other day or kept undisturbed. After delivery, litters from undisturbed dams were submitted to either 180-min daily periods of maternal separations from the 3-14th postnatal days or maintained with the dams all the time. Litters from the stressed dams were left undisturbed from the 3-14th postnatal days. Only males were tested. In adulthood, rats were tested in the elevated T-maze or in the elevated plus-maze. In the latter procedure half the subjects were submitted to a 60-min period of restraint immediately before being tested. The following measures were taken in the elevated plus-maze: frequency and time spent in entries into the arms, stretching, rearing, grooming and head dipping. In the T-maze measures of avoidance and escape latencies were used. Our data indicated that prenatal stress had more pronounced anxiogenic effects than maternal separation, as judged by reduced exploration of the open arms of the elevated plus-maze, but mainly after the restraint stress, and increase in avoidance latencies in the elevated T-maze. The other measures not directly involved in the elevated plus-maze arm exploration yielded similar results. Our data indicate that prenatal stress causes more anxiogenic effects in adulthood than maternal separation but, in the elevated plus-maze, these anxiogenic effects are better seen immediately after an acute stress.

Effect of different illumination levels on rat behavior in the elevated plus-maze.

The present study addressed the role of environmental light intensity on the exploratory behavior of rats in the elevated plus-maze, with the specific goal of determining the light intensity threshold for triggering the aversion to the open arms. Male Wistar-derived rats were tested in the elevated plus-maze under different illumination levels: 0, 1, 3, 10, 30, 100 and 300 lx. Exploratory behavior occurring in the open arms (e.g., entries and time spent in these arms) was more intense under 0 and 1 lx than under the other illumination levels, which did not differ among themselves; on the other hand, locomotor behavior (as indicated by frequency of entries and distance run in the closed arms) was not altered under all illumination conditions. The data indicated that vision is important in triggering aversion to the open arms of the elevated plus-maze. They also indicated that the threshold of such aversion was found between 1 and 3 lx environmental illumination and that the phenomena is not intensity-dependent but rather of an all-or-none type. It should be emphasized that these conclusions only stand for unfamiliar environments. The role of light in familiar environments is currently under investigation in our laboratory.

Dissociation of memory and anxiety in a repeated elevated plus maze paradigm: forebrain cholinergic mechanisms

The effect of intraseptal injection of the cholinergic immunotoxin 192-IgG-saporin on behavior in the elevated plus maze was investigated. A 5-min test-retest paradigm, with minute-by-minute analysis of the first session, was used to evaluate both anxiety and memory in this task. Biochemical analyses revealed a decrease in acetylcholinesterase (AChE) activity in the hippocampus (HPC), septum, and frontal cortex of animals injected with IgG-192 saporin (237.5 ng) when compared with controls. No statistical differences were found between groups in terms of behaviors associated with locomotor activity, conventional measures of anxiety, or ethological behaviors during either session 1 or 2. During test session 2 the controls exhibited decreased exploratory activity and increased indices of anxiety. In contrast, the saporin-treated rats did not exhibit these experience-dependent behavioral changes from session 1 to 2. The minute-by-minute analysis showed a significant decrease in exploratory as well in anxiety associated behaviors during the first session for the control group, but not for the saporin-treated group. These results suggest that the cholinergic innervation of the HPC, the frontal cortex, or both forebrain structures, modulate the initiation of exploratory activity which, results in the acquisition and retention of spatial information, but does not affect the expression of anxiety in the elevated plus-maze.

Behavior ontogeny in the elevated plus-maze: prenatal stress effects.

Prenatal stress is a putative model for studying some psychopathological disorders. Indeed, submitting pregnant animals to stress leads to enhanced anxiety in the adult offspring. However, little is known about how prenatal stress effects interacts with anxiety throughout development. To study this issue, prenatally stressed rats were tested in the elevated plus‐maze at different ages. During pregnancy female rats were submitted to uncontrollable electric foot shock sessions every other day or kept undisturbed (controls). After delivery, litters from control and stressed dams were left undisturbed from the 3rd to the 14th postnatal days. Male and female rats were tested in the elevated plus‐maze at the ages of 30, 45 or 60 days. The following measures were taken in the elevated plus‐maze: number of entries and time spent in the arms (or their extremities) and frequency and time spent in naturalistic behaviors (stretching, rearing, end exploring, grooming and head dipping). Decreases in the percentage of entries into and in the time spent (only females) in the open arms were shown by 60‐day‐old prenatally stressed rats, but not by 30‐ and 45‐day old. Increased open arm ends exploration was shown by 45‐day‐old prenatally stressed males. Rearing behavior was found to increase with age, a phenomenon more pronounced in females. Additionally, at the younger ages prenatally stressed rats were heavier than controls, an effect which disappeared at young adulthood. In conclusion, anxiogenic prenatal stress effects in the elevated plus‐maze could only be detected at early adulthood, not before. Nonetheless, at late adolescence (45 days of age) prenatal stress leaded to an anxiolytic‐like effect which can be interpreted as increased risk‐taking behavior.

Vibrissal sense is not the main sensory modality in rat exploratory behavior in the elevated plus-maze.

Four groups of male Wistar rats were submitted to acute bilateral removal of mystacial vibrissae at different lengths from the follicle. Each group was divided into two subgroups, tested under high (150 Lux) and low environmental illumination (2 Lux). All the subjects were allowed to freely explore an elevated plus-maze for 5 min. Results indicated that rats tested under low illumination tended to explore the open arms more frequently and longer then rats tested under high illumination. When tested under low illumination, rats in the group that suffered whole vibrissa removal stayed longer in the open arms than those in the other groups but did not differ in the number of entries. The average increase in the length of open arm entries, rather than a decrease in aversion to the open arms, may be due to the need of more time to obtain information about the environment since there is no light and the vibrissae were removed. This effect was not seen with rats tested under high illumination, possibly because vision could be used to obtain relevant information.

Effects of apomorphine on rat behavior in the elevated plus-maze

It has been reported that novelty may evoke both an exploratory and a fear drive, thus generating behavior responding to an approach/avoidance conflict. However, not much is known about the approach component. Whereas there exists abundant evidence referring to the avoidance component as the main target for the anxiolytic action of benzodiazepines, the involvement of dopaminergic mechanisms in fear and anxiety is controversial. The present study examined the effects of the dopaminergic agonist apomorphine, the D(2) dopaminergic antagonist sulpiride and the combined treatment sulpiride plus apomorphine on conventional and non-conventional measures of the behavior of rats exposed to an elevated plus-maze. Systemic injection of apomorphine (0.25, 0.5 and 1.0 mg/kg) caused a selective increase in the time spent in the open arms and in the open arm extremities. Pre-treatment with sulpiride blocked these effects while this dopaminergic antagonist had no effect by its own. Apomorphine produced no significant effects on stretching, flat-back-approach or scanning. Therefore, apomorphine increased the behavioral response linked to the approach component of the conflict without affecting risk assessment behaviors. These findings suggest that dopaminergic mechanisms, probably through D(2) receptors, may also be involved in the mediation of the conflict derived from the need of gathering information for confirming, identifying and localizing danger and take the appropriate action for avoiding the threatening stimuli of the elevated plus-maze. A role for dopaminergic mechanisms in the setting up of adaptive responses in a fear-inducing environment is discussed.

Effects of under-and overcrowding on exploratory behavior in the elevated plus-maze

The present work investigated whether the number of rats housed in a cage affects exploration of an elevated plus-maze. Male Wistar-derived rats were kept 1, 2, 3, 4, 6, 8, 12, 16, or 24 to same size cages either for 1 or 14 days and tested in the elevated plus-maze. Rats kept 6 to a cage were arbitrarily considered controls because this is the housing condition adopted in many laboratories, ours included. In comparison to controls, 1-day housed rats kept 1, 2, 16, and 24 to a cage decreased the percentage of entries into the open arms. Similar decreases were also found in the time spent in the open arms, the only exception being the group with rats kept 16 to a cage which failed to show significant differences from the control group. Fourteen-day housed rats kept 1, 2, 16, or 24 to a cage decreased the percentage of entries and time spent in the open arms. We found plus-maze exploration to be similar in groups in which rats were kept from 4 to 12 to a cage. The present data indicate that anxiogenic effects resulting from under- and overcrowding should be taken into consideration in behavioral studies.

The effects of pentylenetetrazol, chlordiazepoxide and caffeine in rats tested in the elevated plus-maze depend on the experimental illumination

The so-called anxiolytic and anxiogenic drugs are considered to cause, respectively, increases and decreases in plus-maze open arm exploration, without modifying locomotor activity occurring in the closed arms in an elevated plus-maze when the animals are tested in an illuminated environment. Simply testing animals in the dark also increases open arm exploration, which may be interpreted as an anxiolytic effect. We investigated the effects of two GABAergic drugs, pentylenetetrazol (10 and 20 mg/kg) and chlordiazepoxide (1.5 and 3 mg/kg), and one non-GABAergic drug, caffeine (10 and 30 mg/kg) on anxiety levels of rats tested in the elevated plus-maze under two illumination conditions, light or dark. All animals explored more the open arms in the dark. In the light, pentylenetetrazol decreased open arm exploration while chlordiazepoxide had the opposite effect. Neither pentylenetetrazol nor chlordiazepoxide had any effect in the dark. Caffeine, increased open arms exploration in both illumination conditions. These results indicate that light triggers aversion, a response mediated by GABA since the GABAergic drugs, but not caffeine, were ineffective when the rats were tested in the dark.

Conflict as a determinant of rat behavior in three types of elevated plus-maze

Three groups of rats were tested in different types of elevated plus-mazes, a normal one (two closed and two open arms), a totally closed one (four closed arms) and a totally open (four open arms). Closed arms were surrounded by 40-cm high wooden walls and open arms were surrounded by 0.5-cm high transparent Plexiglas ledges. As expected, in the closed maze rats explored equally all the arms, both in terms of time and frequency of entries, as well as in exploration of the extremities. Rats in the totally open maze also presented a similar pattern of exploration, that is, no significant differences were found between the results obtained with the closed and the open mazes in terms of central and extremities exploration. It is suggested that the typical behavior of rats in the conventional elevated plus-maze is caused by the contrasting characteristics of open and closed arms rather than by the physical aversive characteristics of the open arms per se. Results also confirm a prediction made by a computer model simulating rat exploratory behavior in virtual mazes, normal, totally open and totally closed.