Rosa M. Escorihuela

Hyperalgesia, anxiety, and decreased hypoxic neuroprotection in mice lacking the adenosine A1 receptor

Caffeine is believed to act by blocking adenosine A1 and A2A receptors (A1R, A2AR), indicating that some A1 receptors are tonically activated. We generated mice with a targeted disruption of the second coding exon of the A1R (A1R−/−). These animals bred and gained weight normally and had a normal heart rate, blood pressure, and body temperature. In most behavioral tests they were similar to A1R+/+ mice, but A1R−/− mice showed signs of increased anxiety. Electrophysiological recordings from hippocampal slices revealed that both adenosine-mediated inhibition and theophylline-mediated augmentation of excitatory glutamatergic neurotransmission were abolished in A1R−/− mice. In A1R+/− mice the potency of adenosine was halved, as was the number of A1R. In A1R−/− mice, the analgesic effect of intrathecal adenosine was lost, and thermal hyperalgesia was observed, but the analgesic effect of morphine was intact. The decrease in neuronal activity upon hypoxia was reduced both in hippocampal slices and in brainstem, and functional recovery after hypoxia was attenuated. Thus A1Rs do not play an essential role during development, and although they significantly influence synaptic activity, they play a nonessential role in normal physiology. However, under pathophysiological conditions, including noxious stimulation and oxygen deficiency, they are important.

A behavioral assessment of Ts65Dn mice : a putative Down syndrome model

Mice which are trisomic for only the human chromosome (Chr) 21-homologous segment of mouse Chr 16 (segmental trisomy), including a portion of the Down syndrome region of human Chr 21, have recently been developed. Since these segmentally trisomic mice, designated Ts(17(16))65Dn, survive to adulthood, they may represent a mouse model for the study of Down syndrome. A partial characterization of their behavioral phenotype was undertaken by evaluating the sensorimotor reflexes, exploration, locomotor activity, emotionality and spatial learning in 16 male Ts65Dn mice (TS) and 16 control (CO) littermates. No sensorimotor deficits appeared in TS compared to CO mice. By contrast, head-dipping behaviour in the hold board was increased in TS mice with respect to the CO group, showing a higher repetition rate of previously explored holes. Crossings in the open field and total arm entries in the plus maze were higher in TS than in the CO group during the dark phase of the light-dark (LD) cycle under red light, but not during the light phase of the LD cycle under white light. Entries into the open arms of the plus maze were increased overall in TS mice when compared to CO mice, but no differences were found in time spent in the open arms. TS mice showed impaired place learning in the Morris water maze, whereas they were able to reach the same performance as CO animals in cued learning. Thus, absence of sensorimotor deficits, increased exploration, hyperactivity under certain experimental conditions and a moderate impairment of spatial learning were the principal characteristics observed in TS mice compared to their CO littermates.

Inbred Roman High- and Low-Avoidance Rats: Differences in Anxiety, Novelty-Seeking, and Shuttlebox Behaviors

In the present study, male inbred animals (from the 10th generation of an inbreeding program that has been carried out in parallel to that of the outbred Roman high- and low-avoidance rat lines), were compared for emotionality in different testing situations, exploratory behavior in the holeboard and two-way, active-avoidance acquisition. Compared to the inbred Roman high-avoidance (RHA-I/Verh) rats, inbred Roman low-avoidance (RLA-I-Verh) rats showed higher emotionality in the open field (reduced distance travelled and number of rearings, and increased self-grooming behavior), in the elevated plus-maze test (increased number of total and open-arm entries, reduced distance travelled in the open arms, and increased self-grooming behavior), and during the habituation period in the shuttle box (decreased number of crossings, increased self-grooming behavior and defecations). Results from the hyponeophagia test were not conclusive, probably due to the test-dependent hyperactivity shown by RHA-I/Verh rats. In the holeboard apparatus, RHA-I/Verh rats explored more than RLA-I/Verh rats, especially when novel objects were located beneath the holes. Finally, RHA-I/Verh animals rapidly acquired active, two-way (shuttlebox) avoidance, whereas RLA-I/Verh animals required four 50-trial sessions to achieve an assymptotic level of 30-40% avoidance. Thus, the behavioral patterns of the Roman inbred strains were very similar to those previously reported for the RHA/Verh outbred lines. Differences in locomotor activity, exploratory, and self-grooming behavior were actually greater between the inbred strains than between the outbred lines. Differences in defecation, however, although still significant, were not so pronounced as those noted previously at this laboratory with the outbred lines.

Neonatal Handling and Environmental Enrichment Effects on Emotionality, Novelty/Reward Seeking, and Age-Related Cognitive and Hippocampal Impairments: Focus on the Roman Rat Lines

Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rats are selected and bred for extreme divergence in two-way active avoidance acquisition. In addition, compared to RLA/Verh rats, RHA/Verh rats are (behaviorally and physiologically) less anxious or reactive to stressors, show increased novelty (sensation)-seeking behavior as well as a higher preference for rewarding substances, and are usually less efficient in learning tasks not involving shock administration. The present article reviews evidence showing that neonatal handling and/or environmental enrichment leads to enduring effects (their magnitude frequently depending upon the rat line) on those behaviors. For example, it has been found that neonatal handling reduces most of the (behavioral and physiological) signs of emotionality/anxiety in RLA/Verh rats, while environmental enrichment increases their novelty seeking (also the case with RHA/Verh rats), saccharin and ethanol intake, and sensitivity to amphetamine. Finally, initial results (currently being further elaborated upon) support a preventive action of both environmental treatments on age-related impairments in learning a spatial, water maze task as well as on hippocampal neuronal atrophy.

Impaired short- and long-term memory in Ts65Dn mice, a model for Down syndrome

Ts65Dn (TS), control littermates (CO) and Swiss (SW) male mice were tested in the elevated plus-maze and in the Morris water maze (MWM) for memory evaluation. In the plus-maze, each mouse was placed at the end of an open arm and initial freezing and the time to enter into an enclosed arm (transfer latency) were measured. SW mice decreased both measures over repeated trials, whereas no decrease of freezing was observed in CO mice, thus suggesting increased emotionality in this group. Compared to CO mice, TS mice showed less initial freezing, shorter transfer latencies, and spent less time in enclosed arms, suggesting a reduced ability to habituate or to inhibit behaviour. Animals were also submitted to a learning-set paradigm consisting of reaching a new platform position each day in the MWM. Two training phases (separated by a resting period of 6 weeks), each including eight acquisition and four cued sessions, were performed (each session consisting of four pairs of trials). CO and SW mice already reached an asymptotic performance by the second day of the first phase whereas TS mice did not achieve that level until the second training phase. The progression over trials indicated that CO and SW animals learned the new platform position between trials 1 and 2 of each session, whereas TS animals failed to do it and had more difficulties to find the platform when it was placed in the centre of the pool as compared to the other positions (SW, NE, E). The results suggest that TS mice show working memory impairments in addition to long-term memory deficits, although extensive training appeared to facilitate TS mice to achieve a level of performance similar to their control littermates. This represents another aspect of the cognitive deficits shown by TS mice: a mouse model of the human Down syndrome.

Mice lacking the adenosine A1 receptor are anxious and aggressive, but are normal learners with reduced muscle strength and survival rate

Behavioural assessment of mice lacking adenosine A1 receptors (A1Rs) showed reduced activity in some phases of the light–dark cycle, reduced exploratory behaviour in the open‐field and in the hole‐board, increased anxiety in the plus maze and dark‐light box and increased aggressiveness in the resident‐intruder test. No differences were found in spatial reference and working memory in several Morris water maze tasks. Both mutant mice had reduced muscle strength and survival rate. These results confirm the involvement of adenosine in motor activity, exploratory behaviour, anxiety and aggressiveness. A1Rs also appear to play a critical role in ageing‐related deterioration.

Genetic selection and differential stress responses - The Roman lines/strains of rats

ETH, Institut fur Natztierwissenschaften, Schorenstrasse 16, CH-8603 Schwerzenbach, Switzerland Autonomous University of Barcelona, Medical Psychology Unit, E-08193 Bellaterra (Barcelona), Spain University of Santiago de Compostela, Department of Psychobiology, E-15705 Santiago de Compostela, Spain University of Cagliari, Department of Toxicology, Viale A. Diaz 182, I-09126 Cagliari, Italy University of Magdeburg, Anatomy Institute, Leipzigerstrasse 44, D-39120 Magdeburg, Germany IUPG, Clinical Psychopharmacology Unit, 100 avenue de Bel-Air, CH-1225 Chene-Bourg (GE), Switzerland NV Organon, RE 2211, P.O. Box 20, NL-5340 BH Oss, the Netherlands University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, GB-Oxford OX3 9DU, England University of Groningen, Department of Animal Physiology, Kerklaan 30, P.O. Box 14, NL-9750 AA Haren, the Netherlands University of Delaware, Departments of Psychology and Biology, Newark, Delaware 19716 USA

The early acquisition of two-way (shuttle-box) avoidance as an anxiety-mediated behavior: Psychopharmacological validation

Several lines of evidence have established that performance during the initial steps of acquisition on a shuttle-box avoidance task is an anxiety-mediated behavior (i.e., the differences between strains selectivity bred for emotionality; the effects of postnatal handling; the course of the corticosterone response and behavioral measures of fear during acquisition). The present study was carried out to add pharmacological evidence to that view by testing the action of anxiogenic and anxiolytic drugs. Single 40-trial sessions with mild shocks (0.4 mA-0.6 mA) were used. In the first experiments the action of sodium pentobarbital (1.25, 2.5 and 5 mg/kg) and three benzodiazepines (diazepam, 2 and 4 mg/kg; alprazolam, 1, 1.25 and 1.5 mg/kg and adinazolam, 1, 2, 4 and 6 mg/kg) were tested. The last two experiments tested a possible proanxiety action of Ro 15-4513 (2, 5 and 10 mg/kg) and FG 7142 (5, 10 and 15 mg/kg), two partial inverse agonists of benzodiazepine receptors, which previous data had suggested to be anxiogenic. The results showed that the measure of acquisition of a two-way active avoidance is a sensitive mean for detecting either anxiolytic or anxiogenic effects of drugs, independently of their effects on locomotor activity, thus suggesting that such test could be a valid model of anxiety in animals.

Effects of postnatal handling of rats on emotional, HPA-axis, and prolactin reactivity to novelty and conflict.

The present studies evaluated whether or not postnatal handling (PH) (administered during the first 21 days of life) could enduringly improve coping behavior with novel and/or conflict situations. To this purpose, different groups of naive male rats (control and PH-treated) were submitted in separate experiments to 1 of the 3 following situations: an emotional reactivity test (in 4-month-old animals), an open-field session followed by endocrine measurements (in 7-month-old animals) and a punished drinking test (in 11-month-old animals). PH effects were significant in the 3 situations: handled animals were less resistant to capture or to the handling manouvers involved in the emotional reactivity test: the hormonal responses (corticosterone, prolactin, and ACTH changes) during and after an open-field stress were less intense, and PH effects lasted up to 11 months in the punished drinking test, as measured by a higher number of punished responses and less time spent freezing by handled animals during the punished period. The results are discussed in relation to previous evidence showing a long-lasting reduction of fearfulness in rats due to postnatal handling.

Enduring effects of environmental enrichment on novelty seeking, saccharin and ethanol intake in two rat lines (RHA/Verh and RLA/Verh) differing in incentive-seeking behavior

The Roman high- and low-avoidance (RHA/Verh and RLA/Verh) rat lines represent, respectively, low emotional/anxious and high novelty seeker vs. high emotional/anxious and low novelty seeker profiles. In the present study, RLA/Verh and RHA/Verh rats, either reared in pairs from weaning (untreated) or reared in groups of 8-10 in an enriched environment until the age of 7 months, were tested for exploratory and novelty-seeking behavior in the hole board (including novel objects under the holes), as well as for their preference for saccharin-water and ethanol-water in a two-bottle free-choice paradigm. Testing started when rats were 20 months old in order to study the long-lasting effects of differential rearing. RHA/Verh rats explored more and showed greater preference for (and intake of) saccharin as well as for ethanol than RLA/Verh rats, thus confirming their validity as a rat model for sensation/reward seeking. Environmental enrichment (EE) increased head-dipping behavior (i.e., novelty seeking) in both rat lines, without affecting locomotor activity. EE treatment increased the preference for, and volume intake of, saccharin (especially at the higher concentrations tested) in the relatively low saccharin-preferring RLA/Verh rats, and also enhanced ethanol consumption in both rat lines. Thus, the results demonstrate consistent and enduring effects of EE on incentive-seeking behavior and further the analysis of how individual differential predispositions for the need of novelty and contact with (or consumption of) rewarding substances arise through either biological (genetic) or early environmental factors, or both.