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Decreased exploratory activity and impaired passive avoidance behaviour in mice deficient for the α1b-adrenoceptor
European Neuropsychopharmacology 10 (2000) 423–427 www.elsevier.com / locate / euroneuro
Decreased exploratory activity and impaired passive avoidance behaviour in mice deficient for the a 1b -adrenoceptor ¨ a,b , * Jens Knauber a , Walter E. Muller b
a Department of Psychopharmacology, Central Institute of Mental Health, Mannheim, Germany Department of Pharmacology, Biocenter, University of Frankfurt Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany
Received 14 December 1999; received in revised form 4 April 2000; accepted 27 June 2000
Abstract There is growing evidence that a dysfunction of central noradrenergic neurotransmission is involved in age-related impairments of cognitive performance and the pathophysiology of Alzheimer’s disease (AD). A reduction of density of central a 1 -adrenergic receptors (a 1 -AR) has been shown in aging and AD brains. Three a 1 -AR subtypes (a 1a , a 1b and a 1d ) have been identified by molecular cloning. However, very little is known about the functional role of distinct a 1 -AR subtypes in the brain. This problem was specifically addressed using a model of knockout mouse deficient in a 1b -AR (a 1B 2 / 2) because these animals show a 40% reduction of a 1 -AR density in the brain as already reported. In comparison to the wild-type mice (a 1B 1 / 1), a 1B 2 / 2 mice showed significantly reduced square entries and a reduced rearing behaviour was observed over all sessions in the open field. In passive avoidance procedures, a 1B 2 / 2 mice showed a tendency towards decreased short-term-latency and a significant decline in long-term-latency. The present results indicate that mutation of a single member of the a 1 -AR gene family creates a distinct phenotype and provide evidence that a 1B -AR is possibly involved in modulation of memory consolidation and fear-motivated exploratory activity. Furthermore, this model of knockout mice may be useful in elucidating the role of a 1B -AR in dementias involving deficits of the noradrenergic system. 2000 Published by Elsevier Science B.V.
1. Introduction The brain noradrenergic system is critically involved in numerous behaviors and behavioral states like arousal, attention, vigilance, anxiety, reactivity to stress or learning and memory processes (Clark et al., 1987; De Sarro et al., 1987; Harley, 1991; Berridge et al., 1993; Sirvio et al., 1994; Abercrombie and Zigmond, 1995; Puumala et al., 1997). It has been speculated that disturbances of number and / or function of a 1 -ARs contribute to cognitive deficits during normal ageing or in different neurological and psychiatric disorders, such as Alzheimer’s disease (AD) and manic-depressive disorders (Moore and Bloom, 1979; Misra et al., 1980; Tomlinson et al., 1981; Brunello et al., 1988; Burnett and Zahniser, 1989; Burnett et al., 1990, 1990; Miyamoto et al., 1990; Lomasney et al., 1991; Sirvio et al., 1994; Pennartz, 1996; Storga et al., 1996; Knauber ¨ and Muller, 2000). Three a 1 -AR subtypes (a 1a , a 1b , and a 1d ) (Bylund and Trendelenburg, 1994) have been iden*Corresponding author.
tified by molecular cloning (Lomasney et al., 1991; Cavallli et al., 1997). However, the functional heterogeneity among a 1 -AR subtypes is not well understood. Unfortunately, the available a 1 -AR specific drugs display only moderate selectivity for different subtypes. For this reason, very little is known about the functional role of distinct a 1 -AR subtypes in brain. In order to further clarify the role of the a 1 -AR and its subtypes for emotional and learning behaviour, a model of knockout mouse deficient of the a 1b -AR (a 1B 2 / 2) was used (Cotecchia et al., 1988; Cotecchia et al., 1990). In this study, the initial behavioral characterisation of a 1B 2 / 2 mice is presented.
2. Materials and methods
2.1. Animals Studies were performed with nine young (4–5 months) male wild-type mice (a 1B 1 / 1) and nine knockout mice (a 1B 2 / 2) deficient for the a 1B -adrenoceptor. The a 1B 2 /
0924-977X / 00 / $ – see front matter 2000 Published by Elsevier Science B.V. PII: S0924-977X( 00 )00100-0
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¨ / European Neuropsychopharmacology 10 (2000) 423 – 427 J. Knauber, W.E. Muller
2 and a 1B 1 / 1 mice were produced and kindly donated from Professor Susanna Cotecchia, Institute of Pharmacology and Toxicology, University of Lausanne. The mice belonged to the F 3 – 5 -generation. Animals were housed in groups of 4–5 per cage, maintained under a 12-h light / dark schedule at a temperature of 228C and allowed free assess to food and water. All behavioral testing procedures have been conducted between 10:00 a.m. and 4:00 p.m. The experimenter was blind to the type of mice investigated.
2.2. Open field behavior Exploration in the open field was carried out in a wooden square arena (50350320 cm) with a floor divided into 16 identical squares of 12.5-cm length. In this procedure the mice were placed individually in the centre of the open field. The pattern and qualitative profile of behavior have been analysed directly by observation. Horizontal activity exploratory activity was measured as the number of squares entered with the forepaws. Vertical activity is represented as number of rears (5rearing up on hind legs). The mice were allowed to freely explore the environment for a 3-min observation period (Q 0 : session 1). Exposure to the box was repeated after 15 min (Q 15min : session 2) and after 24 h (Q 24h : session 3).
3. Results
3.1. Effects of genotype on open field activity 3.1.1. Square entries Multivariate analyses revealed a significant effect of genotype on the number of square entries during three repeated 3-min trials (Fig. 1a) (P,0.02, F57.22, df51) and for both groups a highly significant effect of trial (P,0.0001, F521.21, df52). Comparison of a 1B 2 / 2 and a 1B 1 / 1 mice indicated a highly significant effect of genotype on the 15-min trial (Q 15min ) and the 24-h trial (Q 24h ) (Q 15min : P,0.02, F5 6.92, df51; Q 24h : P,0.02, F57.03, df51). In addition, posthoc analyses of both genotypes indicated a significant decrease of square entries for Q 15min and Q 24h between the a 1B 2 / 2 and a 1B 1 / 1 group (Bonferroni-t-test: t52.12,
2.3. Step-through passive avoidance task The apparatus used consisted of a two-compartment box ¨ as previously described (Knauber and Muller, 2000). The mice were habituated to the apparatus the day before passive avoidance acquisition. On the training trial, the mouse was placed into the lighted chamber and after 15 s the door leading to the dark chamber was opened. When the mouse entered the dark compartment, the door was closed and an inescapable electric foot-shock (0.8 mA, 1-s duration) was delivered from the grid floor. The initial latency (Lnaiv ) to step-through was recorded. The mouse was then removed from the apparatus. The same procedure without the foot-shock was repeated 60 s and 24 h later (L60s and L24h ; cut-off period of 300 s).
2.4. Analyses and statistics Values are means6standard errors of the mean. For statistical analysis (ANOVA, correlation analyses) the SAS package, version 6.04 for DOS was used with group of genotype (a 1B 2 / 2 and a 1B 1 / 1) as the between-subject variable, and the trial latency number (three levels) as the within-subject variable. In the Bonferroni adjustment procedure, the acceptable level of statistical significance (P, 0.05) was divided by the number of pair-wise comparisons. A probability level of less than 0.05 was accepted as statistical significance.
Fig. 1. (a) Effects of genotype on square entries. Q 0 is the initial exploration activity, Q 15min the activity after 15 min, Q 24h the activity after 24 h. ANOVA revealed a highly significant effect of genotype on Q 15min - and Q 24h -trial (Q 15min : P,0.02, F56.92, 1 df; Q 24h : P,0.02, F57.03, 1 df). Posthoc analysis of knockout mice indicated a significant decrease of square entries between the wild-type group and the knockout group (Bonferroni-t-test: t52.12, 16 df, P,0.05). (b) Effects of genotype on rearing. ANOVA revealed a significant effect of genotype on Q 24h -trial (P,0.03, F56.14, 1 df). Posthoc analysis of both genotypes indicated a significant decrease of rears for Q 24h between a 1B 2 / 2 and the a 1B 1 / 1 mice (Bonferroni-t-test: t52.13, 15 df, P,0.05).
¨ / European Neuropsychopharmacology 10 (2000) 423 – 427 J. Knauber, W.E. Muller
df516, P,0,05). For the Q 15min (and Q 24h ) trials, we showed a pronounced decline of the square entries from 9563 (Q 24h : 6762) in the a 1B 1 / 1 mice to 5764 (Q 24h : 4062) in the a 1B 2 / 2 mice. Additionally, there was a tendency towards a difference of the Q 0 trial between a 1B 1 / 1 and a 1B 2 / 2 mice with slightly increased activity of the a 1B 1 / 1 mice, which showed 11765 square entries and, for comparison, a 1B 2 / 2 mice showed only 9563 square entries (P,0.25).
3.1.2. Rearing Multivariate analyses revealed a significant effect of genotype on the number of rears (5sit-ups on hindpaws) during three repeated 3-min trials (P,0.05, F55.69, df5 1). Furthermore, multivariate ANOVA showed for both groups a highly significant effect of trial (P,0.005, F5 6.5, df52). As shown in Fig. 1b, comparison of a 1B 2 / 2 and a 1B 1 / 1 mice indicated a highly significant effect of genotype on Q 24h -trial (P,0.03, F56.14, df51). In addition, posthoc analyses of both genotypes indicated a significant decrease of rears for Q 24h between the a 1B 2 / 2 and the a 1B 1 / 1 group (Bonferroni-t-test: t52.13, df515, P,0.05). There was again a tendency towards a difference of Q 0 and Q 15min (Q 0 : P,0.1; Q 15min : P,0.15) between the a 1B 2 / 2 and the a 1B 1 / 1 group with slightly decreased rearing behaviour of the a 1B 2 / 2 mice, which showed for the Q 0 -trial only 862 rears (Q 15min : 1061 rears), whereas the a 1B 1 / 1 group showed 1561 rears (Q 15min : 1561 rears). 3.2. Effects of genotype on passive avoidance behaviour As shown in Fig. 2, multivariate analysis of this passive-
Fig. 2. Effects of genotype on training (Lnaiv ) and retention latency (L60s , 60 s after stimulus) in a passive avoidance task. ANOVA revealed a highly significant learning effect (P,0.0001, F518.37, 2 df) and significant interaction between learning and genotype (P,0.013, F55.05, 2 df). Posthoc analysis of both genotypes indicated a significant decrease of L24h values between the a 1B 2 / 2 and the a 1B 1 / 1 mice (Bonferroni-ttest: t52.13, 15 df, P,0.05).
425
avoidance task over all genotypes revealed a highly significant effect on passive avoidance performance (P,0.0001, F518.37, df52) and a significant interaction of performance and genotype (P,0.013, F55.05, df52). Furthermore, multivariate ANOVA showed a significant effect of genotype on retention latencies (P,0.02, F57.4, df51). The spontaneous preference for the dark chamber was quite similar in both genotypes with Lnaiv -values of 10.861.2 s in the a 1B 1 / 1 mice and 10.462.5 s in the a 1B 2 / 2 mice, indicating no major alterations of pain sensation. Comparison of both groups indicate a tendency of decreased L60s values from 118.0615.2 s in the a 1B 1 / 1 mice to 42.165.4 s in the a 1B 2 / 2 mice (P,0.16, F5 2.24, df51). In addition, univariate ANOVA showed a highly significant effect of genotype on long trial intervals (L24h ), with pronounced decline of the mean latency of the a 1B 2 / 2 mice to enter the shock chamber 24 h after training as compared to the a 1B 1 / 1 mice (P,0.005, F511.91, df51). L24h values decreased from 224.9611.2 s in the a 1B 1 / 1 mice to 77.368.81s in the a 1B 2 / 2 mice. In this context, posthoc analyses of both genotypes indicated a significant decrease of L24h values between the a 1B 2 / 2 and the a 1B 1 / 1 group (Bonferroni t-test: t5 2.13, df515, P,0.05).
4. Discussion The majority of cognitive behavioral paradigms of mice is confounded by influences of motor activity. For this reason, the first goal was the comparison of exploratory activity of the a 1B 2 / 2 and the a 1B 1 / 1 mice using the open field model to exclude effects of motor activity on more complex learning tasks. In this respect, it could clearly be demonstrated that there were no changes of physical mobility in the a 1B 2 / 2 mice in comparison to the a 1B 1 / 1 mice. This is in accordance with the observation that the a 1B 2 / 2 mice showed normal motor behaviour in their home cage. The more important aspect in the open field model is the simplest measure of mouse emotional behaviour, which is negatively correlated with motor behaviour. Since the open field model is a large and bright arena that is aversive for the nocturnal mice, particularly upon first exposure, physical mobility in this context may also represent emotional / motivational reactivity rather than exploration. Stressful factors are likely to contribute to hypoactivity, like freezing or increased time of immobility as consequences of fear (anxiety). In this respect, the a 1B 2 / 2 mice showed, in contrast to the a 1B 1 / 1 group, low levels of open field activity, as shown by lower initial exploratory activity (square entries) and less numbers of rears in all of the three trials. The a 1B 2 / 2 mice interacted less with the novel object and restricted their movements to the relatively
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¨ / European Neuropsychopharmacology 10 (2000) 423 – 427 J. Knauber, W.E. Muller
sheltered area along the walls of the field. As mentioned before, this reduced motor activity is an index of high emotionality or fear / anxiety of a 1B 2 / 2 mice. Thus, reduced exploration of the open field by mutant animals with a 1B -adrenoceptor deficiency is consistent to heightened responsiveness to stressful environment stimuli (placing in a large novel environment) and in agreement with previous evidence linking NE system function to stress situation (Abercrombie and Zigmond, 1995). Stressinduced release of NE, along with other monoamines, selectively facilitates the neural changes that are required for adaptive behavioural changes. In order to investigate associative learning forms we used passive avoidance procedures in further experiments. In these relatively low-stress situations (prior receiving the footshock) a 1B 2 / 2 and a 1B 1 / 1 mice explored the small test chamber, with the possibility of entry to a dark (preferred) area, in the same fashion. In contrast to the open field model, low stress levels are desired in this task, because high levels of stress are believed to influence cognitive performance. The animals have been habituated to the test apparatus before the testing day and after placing to the test box, the mice soon crossed into the dark chamber. In response to the stress of the training footshock, however, the a 1B 1 / 1 mice altered their behaviour to avoid crossing into the dark chamber while the a 1B 2 / 2 mice did not exhibit avoidance. This impaired retention of the inhibitory avoidance task after receiving a stressful electric stimuli in the a 1B 2 / 2 mice support the idea that an intact NE system i.e. the presence of a 1B -AR is important for learning and cognitive functions, which involves other brain structures than in non-associative learning forms. Other potential explanation cannot be ruled out, since previous studies indicate that the NE system participates in arousal and vigilance. Furthermore, it cannot be completely excluded that both genotypes show different reactivity to footshock because of alterations in sensory detection. Behavioral abnormalities of the a 1B 2 / 2 mice correspond with a significant 42% decline of a 1 -AR density in cerebral cortex and the cerebellum as determined by specific 3 H-prazosin binding two regions with high densities (Cavallli et al., 1997). However, we cannot completely rule out the possibility that additional adaptive mechanisms beside the a 1B -AR loss many contribute to our findings. Therefore, it is important to note that our results of impaired passive avoidance performance of a 1B 2 / 2 mice parallel our previous findings of reduced passive avoidance learning of aged mice also having a reduced density of a 1 -AR in the cerebral cortex of about 31% as determined by specific 3 H-prazosin binding ¨ (Knauber and Muller, 2000). In conclusion, the present results show that mutation of a single member of the a 1 -AR gene family creates a distinct phenotype and provides evidence that a 1B -AR is involved in modulation of memory functions and explorat-
ory activity. Furthermore, this model of the a 1B 2 / 2 mice may be useful in elucidating the role of a 1B -AR in dementias involving deficits of the noradrenergic system. Further procedures are needed to support the hypothesis that emotional and learning behaviour is disturbed in a 1B 2 / 2 mice. The results also support the hypothesis that the central noradrenergic system is critically involved in modulation of emotional and learning behaviour (Berridge et al., 1993; Puumala et al., 1997). Further research is needed to develop a 1 -adrenoceptor agonists and antagonists that demonstrate better selectivity for the different receptor subtypes to assess their effects of interference with central noradrenergic activity in other memory paradigms. The data support the conclusion that the reduction of a 1 -AR density in the aged mouse brain might be critically relevant for the impairment of passive avoidance per¨ formance in aged mice (Knauber and Muller, 2000).
Acknowledgements This study was supported by grants of Dr. W. Schwabe Arzneimittel (Karlsruhe) and Fond der chemischen Industrie (Frankfurt).
References Abercrombie, E.D., Zigmond, M.J., 1995. Modification of central catecholaminergic systems by stress and injury: functional significance and clinical implications. In: Bloom, F.E., Kupfer, D.J. (Eds.), Psychopharmacology: The Fourth Generation of Progress. Raven Press, New York, pp. 355–363. Berridge, C.W., Arnsten, A.F.T., Foote, S.L., 1993. Noradrenergic modulation of cognitive function: clinical implications of anatomical, electrophysiological and behavioural studies in animal models. Psycholog. Med. 23, 557–564. Brunello, N., Riva, M., Rovescalli, A.C., Galimberti, R., Racagni, G., 1988. Age-related changes in rat serotonergic and adrenergic systems and in receptor responsiveness to subchronic desipramine treatment. Pharmacol. Toxicol. 63, 150–155. Burnett, D.M., Zahniser, N.R., 1989. Region-specific loss of a 1 -adrenergic receptors in rat brain with aging: A quantitative autoradiographic study. Synapse 4, 143–155. Burnett, D.M., Daniell, L.C., Zahniser, N.R., 1990. Decreased efficacy of inositol-1,4,5-trisphosphate to elicit calcium mobilisation from cerebrocortical microsomes of aged rats. Mol. Pharmacol. 37, 566–571. Burnett, D.M., Bowyer, J.F., Masserano, J.M., Zahniser, N.R., 1990. Effect of aging on a 1 -adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain. J. Pharmacol. Exp. Ther. 255, 1265–1270. Bylund, D.B., Trendelenburg, U., 1994. International union of pharmacology nomenclature of adrenoceptors. Pharmacol. Rev. 46, 121–134. Cavallli, A., Lattion, A.R. et al., 1997. Decreased blood pressure response in mice deficient of the a 1 -adrenergic receptor. Proc. Natl. Acad. Sci. USA 94, 11589–11594. Clark, C.R., Geffen, G.M., Geffen, L.B., 1987. Catecholamines and attention I. Animal and clinical studies. Neurosci. Behav. Res. 11, 341–352. Cotecchia, S., Schwinn, D.A., Randall, R.R., Lefkowitz, R.J., Caron,
¨ / European Neuropsychopharmacology 10 (2000) 423 – 427 J. Knauber, W.E. Muller M.G., Kobilka, B.K., 1988. Molecular cloning and expression of the cDNA for the hamster a 1 -adrenergic receptor. Proc. Natl. Acad. Sci. USA 85, 7159–7163. Cotecchia, S., Kobilka, B.K., Daniel, K.W., Nolan, R.D., Lapetina, E.Y., Caron, M.G., Lefkowitz, R.J., Regan, J.W., 1990. Multiple second messenger pathways of a 1 -adrenergic receptor subtypes expressed in eukaryotic cells. J. Biol. Chem. 265, 63–69. De Sarro, G.B., Ascioti, C., Froio, F. et al., 1987. Evidence that locus coeruleus is the site where clonidine and drugs acting at a 1 - and a 2 -adrenoceptors affect sleep and arousal mechanisms. Br. J. Pharmacol. 90, 675–685. Harley, C., 1991. Noradrenergic and locus coeruleus modulation of the perforant path-evoked potential in rat dentate gyrus supports a role for the locus coeruleus in attentional and memorial processes. Prog. Brain Res. 88, 307–321. ¨ Knauber, J., Muller, W.E., 2000. Subchronic treatment with prazosin improves passive avoidance learning in aged mice. Possible relationships to a 1 -receptor up-regulation, J. Neural. Transm., in press. Lomasney, J.W., Cotecchia, S., Lefkowitz, R.J., Caron, M.G., 1991. Molecular biology of a 1 -adrenergic receptors: implications for receptor classification and for structure–function relationships. Biochim. Biophys. Acta 1095, 127. Misra, C.H., Shelat, H.S., Smith, R.C., 1980. Effect of age on adrenergic and dopaminergic receptor binding in rat brain. Life. Sci. 27, 521–526.
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Miyamoto, A., Araiso, T., Koyama, T., Ohshika, H., 1990. Membrane viscosity correlates with a 1 -adrenergic signal transduction of aged rat cerebral cortex. J. Neurochem. 55, 70–75. Moore, R.Y., Bloom, F.E., 1979. Central catecholamine systems: anatomy and physiology of the norepinephrine and epinephrine systems. Annu. Rev. Neurosci. 2, 113–168. Pennartz, C.M.A., 1996. The ascending neuromodulatory systems in learning by reinforcement: comparing computational conjectures with experimental findings. Brain Res. Rev. 21, 219–245. ¨ J., 1997. Modulation of vigilance Puumala, T., Riekkinen, Sr. P.J., Sirvio, and behavioral activation by a 1 -adrenoceptors in the rat. Pharmacol. Biochem. Behav. 56, 705–712. Sirvio, J., Lahtinen, H., Riekkinen, Jr. P., Riekkinen, P.J., 1994. Spatial learning and noradrenaline content in the brain and periphery of young and aged rats. Exp. Neurol. 125, 312–315. Storga, D., Vrecko, K., Birkmayer, J.G.D., Reibnegger, G., 1996. Monoaminergic neurotransmitters, their precursors and metabolites in brains of Alzheimer patients. Neurosci. Lett. 203, 29–32. Tomlinson, B.E., Irving, D., Blessed, G., 1981. Cell loss in the locus coeruleus in senile dementia of Alzheimer type. J. Neurol. Sci. 49, 419–428.
Decreased exploratory activity and impaired passive avoidance behaviour in mice deficient for the a 1b -adrenoceptor ¨ a,b , * Jens Knauber a , Walter E. Muller b
a Department of Psychopharmacology, Central Institute of Mental Health, Mannheim, Germany Department of Pharmacology, Biocenter, University of Frankfurt Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany
Received 14 December 1999; received in revised form 4 April 2000; accepted 27 June 2000
Abstract There is growing evidence that a dysfunction of central noradrenergic neurotransmission is involved in age-related impairments of cognitive performance and the pathophysiology of Alzheimer’s disease (AD). A reduction of density of central a 1 -adrenergic receptors (a 1 -AR) has been shown in aging and AD brains. Three a 1 -AR subtypes (a 1a , a 1b and a 1d ) have been identified by molecular cloning. However, very little is known about the functional role of distinct a 1 -AR subtypes in the brain. This problem was specifically addressed using a model of knockout mouse deficient in a 1b -AR (a 1B 2 / 2) because these animals show a 40% reduction of a 1 -AR density in the brain as already reported. In comparison to the wild-type mice (a 1B 1 / 1), a 1B 2 / 2 mice showed significantly reduced square entries and a reduced rearing behaviour was observed over all sessions in the open field. In passive avoidance procedures, a 1B 2 / 2 mice showed a tendency towards decreased short-term-latency and a significant decline in long-term-latency. The present results indicate that mutation of a single member of the a 1 -AR gene family creates a distinct phenotype and provide evidence that a 1B -AR is possibly involved in modulation of memory consolidation and fear-motivated exploratory activity. Furthermore, this model of knockout mice may be useful in elucidating the role of a 1B -AR in dementias involving deficits of the noradrenergic system. 2000 Published by Elsevier Science B.V.
1. Introduction The brain noradrenergic system is critically involved in numerous behaviors and behavioral states like arousal, attention, vigilance, anxiety, reactivity to stress or learning and memory processes (Clark et al., 1987; De Sarro et al., 1987; Harley, 1991; Berridge et al., 1993; Sirvio et al., 1994; Abercrombie and Zigmond, 1995; Puumala et al., 1997). It has been speculated that disturbances of number and / or function of a 1 -ARs contribute to cognitive deficits during normal ageing or in different neurological and psychiatric disorders, such as Alzheimer’s disease (AD) and manic-depressive disorders (Moore and Bloom, 1979; Misra et al., 1980; Tomlinson et al., 1981; Brunello et al., 1988; Burnett and Zahniser, 1989; Burnett et al., 1990, 1990; Miyamoto et al., 1990; Lomasney et al., 1991; Sirvio et al., 1994; Pennartz, 1996; Storga et al., 1996; Knauber ¨ and Muller, 2000). Three a 1 -AR subtypes (a 1a , a 1b , and a 1d ) (Bylund and Trendelenburg, 1994) have been iden*Corresponding author.
tified by molecular cloning (Lomasney et al., 1991; Cavallli et al., 1997). However, the functional heterogeneity among a 1 -AR subtypes is not well understood. Unfortunately, the available a 1 -AR specific drugs display only moderate selectivity for different subtypes. For this reason, very little is known about the functional role of distinct a 1 -AR subtypes in brain. In order to further clarify the role of the a 1 -AR and its subtypes for emotional and learning behaviour, a model of knockout mouse deficient of the a 1b -AR (a 1B 2 / 2) was used (Cotecchia et al., 1988; Cotecchia et al., 1990). In this study, the initial behavioral characterisation of a 1B 2 / 2 mice is presented.
2. Materials and methods
2.1. Animals Studies were performed with nine young (4–5 months) male wild-type mice (a 1B 1 / 1) and nine knockout mice (a 1B 2 / 2) deficient for the a 1B -adrenoceptor. The a 1B 2 /
0924-977X / 00 / $ – see front matter 2000 Published by Elsevier Science B.V. PII: S0924-977X( 00 )00100-0
424
¨ / European Neuropsychopharmacology 10 (2000) 423 – 427 J. Knauber, W.E. Muller
2 and a 1B 1 / 1 mice were produced and kindly donated from Professor Susanna Cotecchia, Institute of Pharmacology and Toxicology, University of Lausanne. The mice belonged to the F 3 – 5 -generation. Animals were housed in groups of 4–5 per cage, maintained under a 12-h light / dark schedule at a temperature of 228C and allowed free assess to food and water. All behavioral testing procedures have been conducted between 10:00 a.m. and 4:00 p.m. The experimenter was blind to the type of mice investigated.
2.2. Open field behavior Exploration in the open field was carried out in a wooden square arena (50350320 cm) with a floor divided into 16 identical squares of 12.5-cm length. In this procedure the mice were placed individually in the centre of the open field. The pattern and qualitative profile of behavior have been analysed directly by observation. Horizontal activity exploratory activity was measured as the number of squares entered with the forepaws. Vertical activity is represented as number of rears (5rearing up on hind legs). The mice were allowed to freely explore the environment for a 3-min observation period (Q 0 : session 1). Exposure to the box was repeated after 15 min (Q 15min : session 2) and after 24 h (Q 24h : session 3).
3. Results
3.1. Effects of genotype on open field activity 3.1.1. Square entries Multivariate analyses revealed a significant effect of genotype on the number of square entries during three repeated 3-min trials (Fig. 1a) (P,0.02, F57.22, df51) and for both groups a highly significant effect of trial (P,0.0001, F521.21, df52). Comparison of a 1B 2 / 2 and a 1B 1 / 1 mice indicated a highly significant effect of genotype on the 15-min trial (Q 15min ) and the 24-h trial (Q 24h ) (Q 15min : P,0.02, F5 6.92, df51; Q 24h : P,0.02, F57.03, df51). In addition, posthoc analyses of both genotypes indicated a significant decrease of square entries for Q 15min and Q 24h between the a 1B 2 / 2 and a 1B 1 / 1 group (Bonferroni-t-test: t52.12,
2.3. Step-through passive avoidance task The apparatus used consisted of a two-compartment box ¨ as previously described (Knauber and Muller, 2000). The mice were habituated to the apparatus the day before passive avoidance acquisition. On the training trial, the mouse was placed into the lighted chamber and after 15 s the door leading to the dark chamber was opened. When the mouse entered the dark compartment, the door was closed and an inescapable electric foot-shock (0.8 mA, 1-s duration) was delivered from the grid floor. The initial latency (Lnaiv ) to step-through was recorded. The mouse was then removed from the apparatus. The same procedure without the foot-shock was repeated 60 s and 24 h later (L60s and L24h ; cut-off period of 300 s).
2.4. Analyses and statistics Values are means6standard errors of the mean. For statistical analysis (ANOVA, correlation analyses) the SAS package, version 6.04 for DOS was used with group of genotype (a 1B 2 / 2 and a 1B 1 / 1) as the between-subject variable, and the trial latency number (three levels) as the within-subject variable. In the Bonferroni adjustment procedure, the acceptable level of statistical significance (P, 0.05) was divided by the number of pair-wise comparisons. A probability level of less than 0.05 was accepted as statistical significance.
Fig. 1. (a) Effects of genotype on square entries. Q 0 is the initial exploration activity, Q 15min the activity after 15 min, Q 24h the activity after 24 h. ANOVA revealed a highly significant effect of genotype on Q 15min - and Q 24h -trial (Q 15min : P,0.02, F56.92, 1 df; Q 24h : P,0.02, F57.03, 1 df). Posthoc analysis of knockout mice indicated a significant decrease of square entries between the wild-type group and the knockout group (Bonferroni-t-test: t52.12, 16 df, P,0.05). (b) Effects of genotype on rearing. ANOVA revealed a significant effect of genotype on Q 24h -trial (P,0.03, F56.14, 1 df). Posthoc analysis of both genotypes indicated a significant decrease of rears for Q 24h between a 1B 2 / 2 and the a 1B 1 / 1 mice (Bonferroni-t-test: t52.13, 15 df, P,0.05).
¨ / European Neuropsychopharmacology 10 (2000) 423 – 427 J. Knauber, W.E. Muller
df516, P,0,05). For the Q 15min (and Q 24h ) trials, we showed a pronounced decline of the square entries from 9563 (Q 24h : 6762) in the a 1B 1 / 1 mice to 5764 (Q 24h : 4062) in the a 1B 2 / 2 mice. Additionally, there was a tendency towards a difference of the Q 0 trial between a 1B 1 / 1 and a 1B 2 / 2 mice with slightly increased activity of the a 1B 1 / 1 mice, which showed 11765 square entries and, for comparison, a 1B 2 / 2 mice showed only 9563 square entries (P,0.25).
3.1.2. Rearing Multivariate analyses revealed a significant effect of genotype on the number of rears (5sit-ups on hindpaws) during three repeated 3-min trials (P,0.05, F55.69, df5 1). Furthermore, multivariate ANOVA showed for both groups a highly significant effect of trial (P,0.005, F5 6.5, df52). As shown in Fig. 1b, comparison of a 1B 2 / 2 and a 1B 1 / 1 mice indicated a highly significant effect of genotype on Q 24h -trial (P,0.03, F56.14, df51). In addition, posthoc analyses of both genotypes indicated a significant decrease of rears for Q 24h between the a 1B 2 / 2 and the a 1B 1 / 1 group (Bonferroni-t-test: t52.13, df515, P,0.05). There was again a tendency towards a difference of Q 0 and Q 15min (Q 0 : P,0.1; Q 15min : P,0.15) between the a 1B 2 / 2 and the a 1B 1 / 1 group with slightly decreased rearing behaviour of the a 1B 2 / 2 mice, which showed for the Q 0 -trial only 862 rears (Q 15min : 1061 rears), whereas the a 1B 1 / 1 group showed 1561 rears (Q 15min : 1561 rears). 3.2. Effects of genotype on passive avoidance behaviour As shown in Fig. 2, multivariate analysis of this passive-
Fig. 2. Effects of genotype on training (Lnaiv ) and retention latency (L60s , 60 s after stimulus) in a passive avoidance task. ANOVA revealed a highly significant learning effect (P,0.0001, F518.37, 2 df) and significant interaction between learning and genotype (P,0.013, F55.05, 2 df). Posthoc analysis of both genotypes indicated a significant decrease of L24h values between the a 1B 2 / 2 and the a 1B 1 / 1 mice (Bonferroni-ttest: t52.13, 15 df, P,0.05).
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avoidance task over all genotypes revealed a highly significant effect on passive avoidance performance (P,0.0001, F518.37, df52) and a significant interaction of performance and genotype (P,0.013, F55.05, df52). Furthermore, multivariate ANOVA showed a significant effect of genotype on retention latencies (P,0.02, F57.4, df51). The spontaneous preference for the dark chamber was quite similar in both genotypes with Lnaiv -values of 10.861.2 s in the a 1B 1 / 1 mice and 10.462.5 s in the a 1B 2 / 2 mice, indicating no major alterations of pain sensation. Comparison of both groups indicate a tendency of decreased L60s values from 118.0615.2 s in the a 1B 1 / 1 mice to 42.165.4 s in the a 1B 2 / 2 mice (P,0.16, F5 2.24, df51). In addition, univariate ANOVA showed a highly significant effect of genotype on long trial intervals (L24h ), with pronounced decline of the mean latency of the a 1B 2 / 2 mice to enter the shock chamber 24 h after training as compared to the a 1B 1 / 1 mice (P,0.005, F511.91, df51). L24h values decreased from 224.9611.2 s in the a 1B 1 / 1 mice to 77.368.81s in the a 1B 2 / 2 mice. In this context, posthoc analyses of both genotypes indicated a significant decrease of L24h values between the a 1B 2 / 2 and the a 1B 1 / 1 group (Bonferroni t-test: t5 2.13, df515, P,0.05).
4. Discussion The majority of cognitive behavioral paradigms of mice is confounded by influences of motor activity. For this reason, the first goal was the comparison of exploratory activity of the a 1B 2 / 2 and the a 1B 1 / 1 mice using the open field model to exclude effects of motor activity on more complex learning tasks. In this respect, it could clearly be demonstrated that there were no changes of physical mobility in the a 1B 2 / 2 mice in comparison to the a 1B 1 / 1 mice. This is in accordance with the observation that the a 1B 2 / 2 mice showed normal motor behaviour in their home cage. The more important aspect in the open field model is the simplest measure of mouse emotional behaviour, which is negatively correlated with motor behaviour. Since the open field model is a large and bright arena that is aversive for the nocturnal mice, particularly upon first exposure, physical mobility in this context may also represent emotional / motivational reactivity rather than exploration. Stressful factors are likely to contribute to hypoactivity, like freezing or increased time of immobility as consequences of fear (anxiety). In this respect, the a 1B 2 / 2 mice showed, in contrast to the a 1B 1 / 1 group, low levels of open field activity, as shown by lower initial exploratory activity (square entries) and less numbers of rears in all of the three trials. The a 1B 2 / 2 mice interacted less with the novel object and restricted their movements to the relatively
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sheltered area along the walls of the field. As mentioned before, this reduced motor activity is an index of high emotionality or fear / anxiety of a 1B 2 / 2 mice. Thus, reduced exploration of the open field by mutant animals with a 1B -adrenoceptor deficiency is consistent to heightened responsiveness to stressful environment stimuli (placing in a large novel environment) and in agreement with previous evidence linking NE system function to stress situation (Abercrombie and Zigmond, 1995). Stressinduced release of NE, along with other monoamines, selectively facilitates the neural changes that are required for adaptive behavioural changes. In order to investigate associative learning forms we used passive avoidance procedures in further experiments. In these relatively low-stress situations (prior receiving the footshock) a 1B 2 / 2 and a 1B 1 / 1 mice explored the small test chamber, with the possibility of entry to a dark (preferred) area, in the same fashion. In contrast to the open field model, low stress levels are desired in this task, because high levels of stress are believed to influence cognitive performance. The animals have been habituated to the test apparatus before the testing day and after placing to the test box, the mice soon crossed into the dark chamber. In response to the stress of the training footshock, however, the a 1B 1 / 1 mice altered their behaviour to avoid crossing into the dark chamber while the a 1B 2 / 2 mice did not exhibit avoidance. This impaired retention of the inhibitory avoidance task after receiving a stressful electric stimuli in the a 1B 2 / 2 mice support the idea that an intact NE system i.e. the presence of a 1B -AR is important for learning and cognitive functions, which involves other brain structures than in non-associative learning forms. Other potential explanation cannot be ruled out, since previous studies indicate that the NE system participates in arousal and vigilance. Furthermore, it cannot be completely excluded that both genotypes show different reactivity to footshock because of alterations in sensory detection. Behavioral abnormalities of the a 1B 2 / 2 mice correspond with a significant 42% decline of a 1 -AR density in cerebral cortex and the cerebellum as determined by specific 3 H-prazosin binding two regions with high densities (Cavallli et al., 1997). However, we cannot completely rule out the possibility that additional adaptive mechanisms beside the a 1B -AR loss many contribute to our findings. Therefore, it is important to note that our results of impaired passive avoidance performance of a 1B 2 / 2 mice parallel our previous findings of reduced passive avoidance learning of aged mice also having a reduced density of a 1 -AR in the cerebral cortex of about 31% as determined by specific 3 H-prazosin binding ¨ (Knauber and Muller, 2000). In conclusion, the present results show that mutation of a single member of the a 1 -AR gene family creates a distinct phenotype and provides evidence that a 1B -AR is involved in modulation of memory functions and explorat-
ory activity. Furthermore, this model of the a 1B 2 / 2 mice may be useful in elucidating the role of a 1B -AR in dementias involving deficits of the noradrenergic system. Further procedures are needed to support the hypothesis that emotional and learning behaviour is disturbed in a 1B 2 / 2 mice. The results also support the hypothesis that the central noradrenergic system is critically involved in modulation of emotional and learning behaviour (Berridge et al., 1993; Puumala et al., 1997). Further research is needed to develop a 1 -adrenoceptor agonists and antagonists that demonstrate better selectivity for the different receptor subtypes to assess their effects of interference with central noradrenergic activity in other memory paradigms. The data support the conclusion that the reduction of a 1 -AR density in the aged mouse brain might be critically relevant for the impairment of passive avoidance per¨ formance in aged mice (Knauber and Muller, 2000).
Acknowledgements This study was supported by grants of Dr. W. Schwabe Arzneimittel (Karlsruhe) and Fond der chemischen Industrie (Frankfurt).
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