Behavioral and neurochemical characterization of α2A-adrenergic receptor knockout mice

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Neuroscience Vol. 113, No. 2, pp. 289^299, 2002 F 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0306-4522 / 02 $22.00+0.00

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BEHAVIORAL AND NEUROCHEMICAL CHARACTERIZATION OF K2A -ADRENERGIC RECEPTOR KNOCKOUT MICE º HDESMA º KI,a J. SALLINEN,b E. MACDONALD,c B. K. KOBILKA,d V. FAGERHOLMa;e and J. LA M. SCHEININa a

Department of Pharmacology and Clinical Pharmacology, University of Turku, Ita«inen Pitka«katu 4, FIN-20520 Turku, Finland b

CNS and Hormone Research and Toxicology, Orion Corporation, Orion Pharma, FIN-20101 Turku, Finland c

d

Department of Pharmacology and Toxicology, University of Kuopio, FIN-70211 Kuopio, Finland

Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, and Division of Cardiovascular Medicine, Stanford University, Stanford, CA 94305, USA e ; bo Akademi University, BioCity, FIN-20520 Turku, Finland Department of Biology, A

Abstract
nerves, smooth muscle contraction, and metabolic and endocrine regulation. The three K2 -AR subtypes (K2A , K2B , and K2C ) are widely distributed both in the CNS and in the periphery (McCune et al., 1993; Scheinin et al., 1994; Nicholas et al., 1996; Wang et al., 1996). The ability to genetically manipulate the K2 -subtypes in mice has provided an approach to elucidate the diverse physiological and behavioral functions of each K2 -AR subtype (for reviews, see MacDonald et al., 1997; Kable et al., 2000; Hein, 2001). The K2C -AR subtype, which is expressed primarily in the CNS (striatum, olfactory tubercle, hippocampus and cerebral cortex), has an inhibitory function in the modulation of several aspects of mouse behavior, such as startle reactivity, aggressive behavior, and amphetamine-induced locomotor hyperactivity. In addition, the K2C -AR also has a role in modulating responses in mouse models assessing learning and memory as well as depression. In contrast, the K2B -AR subtype shows mainly peripheral expression (kidney, liver, lung, heart and vascular tissues), and is responsible for the immediate hypertensive response to intravenously administered K2 -agonists through peripheral vasoconstriction. The K2A -AR is the most abundant K2 -AR subtype expressed

The K2 -adrenergic receptors (K2 -AR) mediate a variety of physiological responses and pharmacological e¡ects in the CNS, mainly by inhibiting neuronal ¢ring and release of norepinephrine (NE) and other neurotransmitters. Within the CNS, K2 -AR participate in the mechanisms that regulate blood pressure, antinociception, body temperature, cognitive functions and mood. In the periphery, K2 -AR are also involved in multiple functions, including modulation of NE release from sympathetic

*Corresponding author. Tel.: +358-2-3337502; fax: +358-23337216. E-mail address: mika.scheinin@utu.¢ (M. Scheinin). Abbreviations : 5-HIAA, 5-hydroxyindoleacetic acid ; 5-HT, 5-hydroxytryptamine or serotonin; K2A -KO, mice lacking the K2A AR ; ANOVA, analysis of variance ; AR, adrenergic receptor; DA, dopamine; D79N mice, mice with dysfunctional K2A -AR; DOPAC, 3,4-dihydroxyphenylacetic acid ; HVA, homovanillic acid ; MHPG, 3-methoxy-4-hydroxyphenylglycol ; NE, norepinephrine; RS-79948-197, [8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a, 13,13a-decahydro-12-(ethylsulfonyl)-3-methoxy-6H-isoquino[2,1-g][1,6]naphthyridine hydrochloride; TRP, tryptophan ; WT, control mice for mice lacking the K2A -AR. 289

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widely both in the CNS and in peripheral tissues. In the CNS, mRNA for the K2A -AR is found in the locus ceruleus and in other noradrenergic cell body regions, and also in other brainstem centers controlling sympathetic out£ow. In addition, the K2A -AR is highly expressed in all regions of cerebral cortex, in the hippocampus, in the septum, in most hypothalamic and amygdaloid nuclei, and in the spinal cord (Wang et al., 1996). Experiments on mice with targeted inactivation of the gene for K2A -AR (K2A -knockout, K2A -KO) or with a transgene encoding a dysfunctional receptor protein (K2A -D79N), although not totally converging, have revealed distinct phenotypic alterations in these mutant mouse strains, including tachycardia, elevated blood pressure, elevated plasma NE levels, increased NE turnover in hippocampus, and a lowered threshold for seizures in the kindling model of epilepsy (Lakhlani et al., 1997; Janumpalli et al., 1998; Altman et al., 1999; Makaritsis et al., 1999). The K2A -AR has also emerged as the main mediator of presynaptic K2 -adrenergic inhibition of NE release in the heart atria, vas deferens, cerebral cortex and hippocampus, although also K2C -AR participates in this feedback regulation especially at low stimulation frequencies (Altman et al., 1999; Hein et al., 1999; Trendelenburg et al., 1999, 2001). Moreover, in pharmacological experiments, most of the classical e¡ects of K2 -AR agonists have been attributed to the K2A -AR subtype; mutation or deletion of the K2A -AR leads to loss of the hypotensive, antinociceptive, sedative^hypnotic, anesthetic-sparing, and hypothermic responses to K2 -agonists (MacMillan et al., 1996; Hunter et al., 1997; Lakhlani et al., 1997; Altman et al., 1999). Transgenic and gene-targeting techniques with mice are increasingly used to study human gene function. A mouse model of human gene function may, however, be of limited value until proper phenotypic assessments are performed. Indeed, di¡erent guidelines for the analysis of the behavioral phenotypes of new transgenic and knockout mouse strains have recently been introduced (Crawley and Paylor, 1997; Rogers et al., 1997). The analysis of basic neurologic function in these test batteries is based on the primary screen described by Irwin (1968) that has been widely used for screening drug candidates in pharmaceutical laboratories. These standardized models provide a behavioral and functional pro¢le of a mutant mouse strain and thus form a valuable basis for the application of further, more speci¢c, hypothesis-driven behavioral paradigms. The aim of the current experiments was to characterize the phenotype of mice with targeted inactivation of the gene for K2A -AR, employing a behavioral characterization test battery. The primary behavioral screen was used to provide a behavioral and functional pro¢le of the knockout mouse strain, because such characterization has not been previously reported in K2A -KO mice. The primary screen revealed that the K2A -KO mice exhibited alterations in anxiety-related behaviors. Therefore, the mice were further analyzed in di¡erent simple models of anxiety, i.e. the open ¢eld test, the marble burying test, and the elevated-plus maze test. Neurochemical

analysis was carried out to determine the in£uence of the absence of the main presynaptic K2 -AR subtype on metabolism of NE in di¡erent brain regions. The objective of the neurochemical assessment also included an evaluation of whether the lack of K2A -AR would have an in£uence on the metabolism of other monoamine neurotransmitters, dopamine (DA) and serotonin (5-HT). In addition, to obtain an overview of the remaining CNS K2 -AR binding sites in K2A -KO mice, receptor autoradiography of the brain was performed using the subtype-non-selective K2 -AR antagonist radioligand [3 H][8aR,12aS,13aS]-5,8,8a,9,10,11,12,12a,13,13a-decahydro-12-(ethylsulfonyl)-3-methoxy-6H-isoquino[2,1-g][1,6]naphthyridine hydrochloride [3 H]RS-79948-197.

EXPERIMENTAL PROCEDURES

Experimental animals A total of 128 male mice of 10^22 weeks of age, and 20 female mice of 20^36 weeks of age were used. The generation of an K2A -KO mouse strain has been described previously (Altman et al., 1999). K2A -KO mice were backcrossed for ¢ve generations to C57BL/6J mice to form a congenic strain. Age-matched wildtype C57BL/6J (wild-type, WT) mice of the same genetic background (Jackson Laboratories, Bar Harbor, ME, USA) were used as control animals. All e¡orts were made to minimize animal su¡ering and the number of animals used. All experiments conformed to the European Communities Council Directive 86/ 609/EEC, and the study had the approval of the local committee for laboratory animal welfare. The mice were housed under standard laboratory conditions with a 12-h light/dark cycle (lights on at 6.00 h and o¡ at 18.00 h). Experiments were conducted between 08.00 and 16.30 h. Receptor autoradiography Male mice of 12^14 weeks of age were decapitated under light CO2 anesthesia. Brains were rapidly removed and frozen by immersion in isopentane chilled in an ethanol/dry-ice slurry. Brain sections, 14 Wm thick, were cut on a cryostat and thawmounted onto gelatinized glass slides. Brain sections were labeled with the K2 -AR antagonist radioligand [3 H]RS-79948-197 (Amersham Pharmacia Biotech, Buckinghamshire, UK). Sections were incubated with 0.5 nM [3 H]RS-79948-197 in 50 mM potassium phosphate bu¡er, pH 7.4, for 1 h at room temperature. Non-speci¢c binding was de¢ned as [3 H]RS-79948-197 binding in the presence of 10 WM phentolamine. After washing in fresh ice-cold bu¡er (2 s+20 min), the slides were dipped in ice-cold deionized water to remove salts, and dried in a stream of air. The dry slides were apposed to Kodak BioMax MR autoradiographic ¢lm for 16 weeks. These experimental conditions have been validated and described in detail elsewhere (Fagerholm and Scheinin, submitted). Autoradiographic images were digitized using a CCD video camera. Primary behavioral screen Twenty-one K2A -KO and 18 WT male mice of 18^22 weeks of age were used for this test. The employed battery of behavioral observational tests was a modi¢cation of the Irwin procedure (Irwin, 1968), during which a total of 40 separate measurements are recorded for each animal (for a full method description, see Rogers et al., 1997). Blind assessment of each animal began with observation of undisturbed behavior in a transparent cylindrical viewing jar (11 cm in diameter). The following behaviors were scored during 5 min in the viewing jar:

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Body position: completely £at (0) to repeated vertical leaping (8) Spontaneous activity: none (0) to repeated vigorous movement (6) Piloerection: none (0) or marked (1) Respiration: gasping, irregular (0) to hyperventilation (3) Tremor: none (0) to marked (2) The mice were then transferred to an arena (55U33 cm) that consisted of 15 even-sized squares (11U11 cm) for observation of the following behaviors assessing the immediate reactions of the animal to a new environment: Transfer arousal: coma (0) to extremely excited (6) Locomotor activity: number of squares entered by all four feet in 30 s Palpebral closure: eyes wide open (0) to eyes closed (2) Gait: normal (0) to incapacity (3) Pelvic elevation: £attened (0) to elevated more than 3 mm (3) Tail elevation: £attened (0) to elevated (2) Startle reaction: the jerk produced in response to a pen hitting against the Perspex arena wall ^ no response (0) to 1 cm jump or more (3) Touch escape: no response (0) to extremely vigorous (3) This was followed by a sequence of manipulations using tail suspension and observation on a wire grid: Visual placing: animal lowered and distance from grid needed to evoke forepaw paddling ^ none, i.e. with nose in contact (0) to 25 mm or more (3) Trunk curl: absent (0) or present (1) Limb grasping: absent (0) or present (1) Grip strength: resistance when animal placed on grid and drawn backwards ^ none (0) to unusually e¡ective (4) Ear re£ex: ear retraction in response to a light pu¡ of air ^ none (0) to repetitive £ick (3) Corneal re£ex: blink response to a light pu¡ of air ^ none (0) to repeated eye blink (3) Toe pinch: none (0) to very brisk with repeated extension and £exion (4) Wire maneuver: animal suspended from horizontal wire by forelimbs and released ^ active and grasps with hind limbs (0) to falls immediately (4) Body tone: determined by compression of the sides with thumb and fore¢nger ^ completely £accid (0) to extreme resistance (2) Contact righting re£ex: animals placed in a plastic tube (diameter 33 mm) and turned upside down ^ normal quick turn (0) to not turning (2) The animals were then restrained in a supine position using tail-neck grip and the following phenomena were scored: Struggle to tail-neck grip: absent (0) or present (1) Heart rate: palpated below sternum ^ lowered (0) to elevated (2)

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Provoked biting response: absent (0) or present (1) Limb tone: ¢nger gently pressed against the plantar surface of each hindpaw ^ no resistance (0) to extreme resistance (4) Abdominal tone: abdomen gently palpated with ¢nger ^ completely £accid (0) to extreme resistance (2) Immediately thereafter, core body temperatures were measured using a rectal probe and a digital thermometer (Ellab, Roedovre, Denmark). The probe was inserted 2.5 cm inside the anal sphincter. As the last procedure, motor coordination of the mice was analyzed using the rotarod test (Jones and Roberts, 1968). Mice were placed individually on the rotating drum (Ugo Basile, Comerio, Italy). The drum rotated with accelerating speed for a maximum of 5 min, and the time at which each animal fell from the drum was recorded. Spontaneous locomotor activity Spontaneous locomotor activity of K2A -KO and WT mice (sixteen 18^22 week-old male mice per group) was measured by placing the animals individually into polypropylene animal cages (38U22U15 cm) with transparent polypropylene lids and an approximately 1.5-cm-thick layer of granulated aspen bedding on the £oor. The cages were surrounded with a photobeam frame system designed for activity measurements (Photobeam Activity System PAS, Cage Rack, San Diego Instruments, San Diego, CA, USA). The system consisted of 16 separate frames connected to a computer control unit. The system enabled the simultaneous measurement of 16 mice at two levels (3 and 6 cm from the bottom of the cage). Three di¡erent variables were recorded: (1) ambulations (breaks of two adjacent beams of light at the lower level), (2) ¢ne movements (two consecutive breaks of a single beam of light at the lower level), and (3) rearings (one break of a beam of light at the upper level). The locomotor activity was measured over 1-h intervals for 24 h. The measurements started at 12.00 h, after 2 h habituation of the mice in the recording cages. Elevated-plus maze test The apparatus for the elevated-plus maze test (Lister, 1987) comprised two open arms (30U5U0.3 cm) facing each other and two closed arms (30U5U15 cm). The arms extended from a common central platform (5U5 cm), and the entire apparatus was elevated to a height of 50 cm above £oor level. The maze £oor was constructed of black Plexiglas and the side and end walls of the closed arms were constructed of clear Plexiglas. A small lip (0.3 cm), as described by Cole and Rodgers (1994), was used in the open arms to prevent the mice from falling from the maze. Nineteen K2A -KO and 20 WT male mice of 11^13 weeks of

Table 1. Summary of the di¡erences between the K2A -KO and WT mice observed in the primary behavioral screen A

Number of animals exhibiting the behavior

P level (Pearson’s M2 test)

WT n = 18

K2A -KO n = 21

Piloerection marked Tail elevated Grip struggling present Heart rate increased Provoked biting response present Contact righting re£ex impaired

0 (0%) 2 (11%) 18 (100%) 4 (22%) 18 (100%) 7 (39%)

5 (24%) 9 (43%) 3 (14%) 13 (62%) 3 (14%) 16 (76%)

B

Mean U S.E.M. of the observed behavior

P level (t-test)

Number of crossings in open arena Rotarod latency (s)

19.8 U 0.9 187 U 16

P = 0.008 P 6 0.001

15.9 U 1.1 111 U 14

P = 0.027 P = 0.028 P 6 0.001 P = 0.034 P 6 0.001 P = 0.045

The results for categorical variables (A) are presented as number of animals exhibiting each behavior and for continuous variables (B) as means U S.E.M.

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Fig. 1. Autoradiographic images of horizontal (A, B) and sagittal (C, D) brain sections of WT (A, C) and K2A -KO (B, D) mice labeled with the K2 -adrenoceptor antagonist [3 H]RS-79948-197 (0.5 nM). Binding in K2A -KO mice was greatly reduced in most, but not all brain regions. Scale bar = 2 mm.

age were used for the elevated-plus maze test. Each mouse was placed in the center of the maze facing an open arm. During the 5-min test the number of entries into each arm, the time spent in each arm type and the number of rearings and exploratory head dips were recorded. A mouse was deemed to have entered an arm when all four legs were on the arm. The maze was cleaned with wet and dry paper towels after each mouse. The tester was blind to the genotype of the mice.

Open ¢eld mobility test The apparatus for the open ¢eld test was the same as the one used in the measurement of spontaneous locomotor activity (see above). Female K2A -KO (n = 10) and WT (n = 10) mice (20^36 weeks of age) were placed individually in locomotor activity measurement cages 30 min after a s.c. injection of saline (5 ml/kg). During a 2-h measurement time, ¢ne movements, ambulations and rearings were recorded.

Marble burying test Analysis of brain monoamines and their metabolites The marble burying test was performed in standard polypropylene laboratory animal cages (38U22U15 cm), in which 24 glass marbles (diameter 1.2 cm) were spaced evenly on the aspen bedding. Mice were placed individually in a cage and left undisturbed for 30 min. After the test, the number of marbles buried (covered with bedding more than two thirds of the marble size) was counted. The subjects were 10 K2A -KO and 10 WT male mice aged 10^11 weeks.

Eight K2A -KO and eight WT male mice (aged 17^22 weeks) were decapitated, and brains were rapidly removed. Cerebral cortex, hippocampus, striatum and the thalamus and hypothalamus were dissected and placed in preweighed tubes on dry ice. Biogenic amines (NE, DA, 5-HT) and their metabolites were determined from brain homogenates in 0.1 M perchloric acid using electrochemical detection (ESA Coulchem, 5011, Bedford,

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Fig. 2. Undisturbed locomotor activity of K2A -KO (n = 16) and WT (n = 16) mice was measured for 24 h over 1-h intervals starting at 12.00 h, after a 2-h habituation. The total activity (ambulations, ¢ne movements and rearings; mean+S.E.M.) of the K2A -KO mice during the dark period (from 18.00 h to 06.00 h) was markedly attenuated compared to the WT animals (P 6 0.001). Asterisks refer to signi¢cant di¡erences in t-tests between genotypes at di¡erent time points. *P 6 0.05; **P 6 0.01; ***P 6 0.001.

MA, USA) after separation by high-performance liquid chromatography on a reversed-phase C18 column (Ultrasphere ODS, 4.6U250 mm, Beckman Instruments, Fullerton, CA, USA). The bu¡er systems described by Me¡ord (1981) were used, with the minor modi¢cations described elsewhere (MacDonald et al., 1988). Data analysis The results are presented as means U S.E.M., or as frequencies (primary behavioral screen). Statistical analysis was carried out using SPSS for Windows 8.0.1 (SPSS Inc., Chicago, IL, USA). Pearson’s M2 test was used to analyze data from the primary behavioral screen (categorical variables). Continuous variables from the primary behavioral screen as well as data from the elevated-plus maze test and neurochemical assessments were evaluated with independent-samples t-tests. Data from the marble burying test were analyzed using Mann^Whitney’s U-test. Comparisons of locomotor activity measurements, as well as the data from the open ¢eld test, were performed using twoway analysis of variance (ANOVA) for repeated measurements. Alpha was set at 0.05.

RESULTS

Receptor autoradiography Compared to WT mice, [3 H]RS-79948-197 binding was greatly reduced in most brain regions of K2A -KO

mice (Fig. 1B, D). In WT mice, [3 H]RS-79948-197 binding was seen in both K2A - and K2C -AR-expressing brain structures (Fig. 1A, C). In the brain sections of the K2A KO mice, the highest binding densities were observed in the caudate^putamen, the nucleus accumbens, the olfactory tubercle, the reticular thalamic nucleus, substantia nigra, the cerebellar nuclei and the hippocampus (Fig. 1B, D). Behavioral characterization of mice lacking K2A -AR Primary behavioral screen. The targeted inactivation of the gene encoding the K2A -AR was associated with signi¢cant di¡erences in comparison with WT control animals in the primary behavioral screen in the following measures summarized in Table 1. Piloerection was observed exclusively among K2A -KO mice (¢ve out of 21; P = 0.027). After transfer to an open arena, K2A KO mice had reduced locomotor activity in the novel environment (P = 0.008) and a higher frequency of elevated tail (nine out of 21) compared to WT mice (two out of 18; P = 0.028). In the supine position, K2A -KO mice were less likely to be provoked into biting (three out of 21) than control animals (18 out of 18; P 6 0.001), and they more often had increased heart rate (13 out of 21) than the WT control mice (four out of 18; P = 0.034).

Table 2. Elevated-plus maze test performance of K2A -KO and WT mice

Time in open arms (s) Closed entries (count) Open entries (count) Rearings (count) Head dips (count)

WT n = 20

K2A -KO n = 19

P level (t-test)

76 U 17 7.5 U 0.7 4.4 U 0.7 6.1 U 0.7 3.5 U 1.1

30 U 6 10.4 U 0.6 2.7 U 0.5 7.8 U 0.6 0.7 U 0.3

P = 0.024 P = 0.003 NS (P = 0.064) NS (P = 0.054) P = 0.032

Data are means U S.E.M.

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1.28 U 0.04 1.32 U 0.06 3.00 U 0.10 2.84 U 0.20 2.14 U 0.07 2.18 U 0.14 5.16 U 0.09 5.26 U 0.35 2.14 U 0.06 2.31 U 0.08 3.23 U 0.15 2.99 U 0.17 2.92 U 0.21 3.31 U 0.14 7.42 U 0.12 7.94 U 0.24 14.6 U 0.5 16.8 U 1.4 11.8 U 0.6 13.1 U 0.9 15.9 U 0.7 18.0 U 1.4 15.2 U 0.5 16.5 U 1.6 Thalamus+ hypothalamus

Striatum

Hippocampus

WT KO WT KO WT KO WT KO Cortex

Values are expressed as means U S.E.M. In the K2A -KO mice, an increase in NE turnover was observed in all regions. Asterisks refer to signi¢cant di¡erences in t-tests between genotypes (n = 8 per genotype group). *P 6 0.05; **P 6 0.01; ***P 6 0.001.

1.22 U 0.10 1.29 U 0.13 0.58 U 0.07 0.65 U 0.07 0.09 U 0.00 0.08 U 0.00 0.63 U 0.01 0.61 U 0.03 0.57 U 0.04 0.63 U 0.05 0.59 U 0.07 0.63 U 0.12 0.05 U 0.00 0.05 U 0.00 0.39 U 0.01 0.41 U 0.01 0.53 U 0.04 0.56 U 0.06 0.18 U 0.00 0.20 U 0.01 5.85 U 0.26 5.60 U 0.30 1.17 U 0.03 1.09 U 0.07* 0.24 U 0.02 0.27 U 0.02 0.19 U 0.01 0.19 U 0.02 3.22 U 0.10 3.27 U 0.20 0.71 U 0.02 0.73 U 0.02 0.43 U 0.00 0.44 U 0.01 0.35 U 0.04 0.33 U 0.03 62.8 U 3.7 67.1 U 2.7 1.84 U 0.05 1.77 U 0.06 0.14 U 0.02 0.32 U 0.03*** 0.11 U 0.01 0.16 U 0.02* ND ND 0.09 U 0.01 0.16 U 0.02** 0.13 U 0.01 0.30 U 0.03*** 0.21 U 0.02 0.30 U 0.03* ND ND 0.16 U 0.01 0.31 U 0.02***

DOPAC/DA HVA/DA HVA DOPAC DA MHPG/NE MHPG NE

0.99 U 0.08 0.95 U 0.03 2.02 U 0.12 1.91 U 0.08 ND ND 1.85 U 0.17 2.06 U 0.19

5-HIAA NSC 5630 11-7-02

TRP

5-HT

Elevated-plus maze test. In the elevated-plus maze test, the K2A -KO animals spent less time in the open arms of the maze than the WT mice (Table 2). The

Genotype

Locomotor activity measurement. The 24-h locomotor activity measurement (Fig. 2, total activity) revealed that the K2A -KO mice had a di¡erent diurnal activity pattern as compared to the WT controls. ANOVA showed a signi¢cant genotypeUtime interaction (P = 0.001). Further analyses were performed where the dark (from 18.00 to 06.00 h) and light periods were tested separately. The increase in activity during the dark period was blunted in K2A -KO mice compared to WT controls (P 6 0.001). On the other hand, during the light period, the variation seen in locomotor activity was only dependent upon time (e¡ect of time, P 6 0.001), whereas no e¡ect of the genotype (P = 0.16) was observed. The di¡erence was similar in all components of locomotor activity (results not shown).

Brain region

Impaired contact righting re£ex was also more frequently observed among K2A -KO mice (16 out of 21) than in WT mice (seven out of 18; P = 0.045). In the rotarod test, K2A -KO mice had shorter latency to fall from the rotating bar in comparison with WT animals (P 6 0.001). No signi¢cant di¡erences (P s 0.15) were detected in the following variables: body position, spontaneous activity in the viewing jar, respiration, transfer arousal, pelvic elevation, startle reaction, touch escape, visual placing, grip strength, ear re£ex, corneal re£ex, toe pinch, wire maneuver, limb tone, and abdominal tone. No deviations from normal were observed in either K2A KO or WT animals in tremor, palpebral closure, gait, trunk curl, limb grasping and body tone. Body temperature was similar in both strains (WT, 38.0 U 0.1‡C; KO, 37.9 U 0.1‡C).

Table 3. Monoamines and their metabolites (nmol/g of tissue) in di¡erent brain regions of K2A -KO and WT mice

Fig. 3. Marble burying behavior of individual K2A -KO and WT mice. The K2A -AR-de¢cient mice buried signi¢cantly fewer glass marbles during the 30-min test (P 6 0.001). Horizontal lines represent medians of each genotype (n = 10 in each genotype).

0.60 U 0.03 0.57 U 0.02 0.94 U 0.04 0.95 U 0.05 0.75 U 0.04 0.66 U 0.03 0.70 U 0.02 0.66 U 0.03

J. La«hdesma«ki et al. 5-HIAA/5-HT

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Fig. 4. Rearing behavior (mean U S.E.M.) of female K2A -KO (n = 10) and WT (n = 10) mice in the open ¢eld test. Rearing behavior of the K2A -KO mice was decreased compared to WT animals (P = 0.034).

K2A -KO mice also tended to have fewer open arm entries and more rearings, but the di¡erences did not reach statistical signi¢cance. Furthermore, the K2A -KO mice performed fewer head dips, and more entries into closed arms. No statistically signi¢cant di¡erences between the two genotypes were observed in the total number of arm entries. Marble burying test. In the marble burying test, the K2A -KO mice buried markedly fewer glass marbles compared to WT mice (KO, 6.3 U 2.4; WT, 18.8 U 1.4) (P 6 0.001) (Fig. 3). Open ¢eld test. Among the measured behaviors, a signi¢cant e¡ect of genotype was detected only in rearings (P = 0.034), the K2A -KO mice having fewer rearings than WT animals (Fig. 4). There were no signi¢cant differences between the genotype groups in ¢ne movements or ambulations (P = 0.22 and 0.29, respectively) (results not shown). Brain biogenic amines and their metabolites Lack of K2A -AR expression was associated with signi¢cantly elevated levels of 3-methoxy-4-hydroxyphenylglycol (MHPG), the principal metabolite of NE, throughout the studied brain regions (cortex MHPG +231% compared to WT) (P 6 0.001) (Table 3). In addition, in the K2A -KO mice the MHPG/NE ratio was also increased in all brain regions (cortex MHPG/NE ratio +229% compared to WT) (P 6 0.001). No marked di¡erences were detected in the levels of DA, 5-HT or their metabolites.

DISCUSSION

Receptor autoradiography with the subtype-non-selective K2A -AR antagonist radioligand [3 H]RS-79948-197 con¢rmed the lack of functional K2A -AR protein in the K2A -KO mouse strain. In K2A -KO mice, the distribution of [3 H]RS-79948-197 binding sites resembled that observed with the K2C - over K2A -AR-preferring ligands [125 I]rauwolscine-OHPC and [3 H]rauwolscine in mice (Dossin et al., 2000) and rats (Boyajian et al., 1987), suggesting that the [3 H]RS-79948-197 binding in K2A KO mice indeed represents binding to non-K2A -AR. More comprehensive analysis of K2 -AR binding in the CNS of K2A -KO mice has indicated that no signi¢cant compensatory up-regulation of other K2 -AR subtypes takes place, since no increase in labeling of K2C -AR with [3 H]rauwolscine was evident in K2A -KO compared to WT mice (Fagerholm and Scheinin, submitted). Although the phenotypes of K2A -KO and WT mice were generally indistinguishable in normal housing conditions, detailed scrutiny revealed a number of di¡erences between the mutant mice and their controls. Here, we have listed the main ¢ndings of the study and a more detailed discussion of each result will be given later in this section. The K2A -AR gene disruption was associated with an increased frequency of piloerection, elevated heart rate, and Straub tail ( = elevated tail) in the primary behavioral screen of the mouse strain. The K2A -KO mice also seemed to be more vulnerable to environmental stressors, such as being introduced into a novel environment. Moreover, the K2A -AR-de¢cient mice had impaired contact righting re£ex and rotarod performance. The K2A -KO mice exhibited increased anxiety-like behav-

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ior in the elevated-plus maze test as well as reduced exploratory behavior in the open ¢eld test. In comparison with the WT mice, the diurnal activity pattern of K2A -KO mice was altered in the 24-h locomotor activity test: the normal increase in locomotor activity during the dark phase was clearly blunted. In addition, the turnover of NE was markedly increased in the K2A -KO mice in all studied brain regions, whereas no di¡erences in the metabolism of other monoamines were detected between the genotypes. The increased frequency of piloerection observed in K2A -KO mice may have resulted from increased sympathetic nervous system activity (Hein et al., 1999) or increased anxiety, observed in the current experiments and also in a recently published study employing the same K2A -KO mouse strain (Schramm et al., 2001). The elevation of heart rate of K2A -KO mice, here observed simply by substernal palpation, has also been previously veri¢ed in anesthetized animals using arterial recording (Altman et al., 1999). It is likely that the tachycardia in the K2A -KO mice results primarily from increased sympathetic tone caused by the loss of K2A AR-mediated inhibition of the vasomotor center together with the loss of K2A -AR-mediated inhibition of NE release from peripheral cardiac nerve terminals (Altman et al., 1999). Decreases in both grip struggling and provoked biting behavior of K2A -KO mice may re£ect an exaggerated stress reaction to the slightly or moderately stressful experimental conditions, and/or attenuated defensive responsivity. This was somewhat unexpected since the hypernoradrenergic state of K2A -KO mice would be expected to increase rather than decrease aggressive behavior. However, it is likely that in K2A -KO mice the increase in sympathetic activity during a period of stress or arousal is not followed by the normally occurring autoinhibition that would limit the noradrenergic over£ow caused by the ‘¢ght or £ight’ response. Previously, increased aggression has been observed in mice lacking the K2C -AR subtype, whereas overexpression of K2C -AR was associated with decreased aggression in the isolationinduced aggression test (Sallinen et al., 1998). After transfer to an open arena, the K2A -KO mice now ‘froze’ for a longer period of time than the WT mice before starting to move in the novel environment, as evidenced by the reduced number of crossings during the ¢rst 30 s after the transfer. This might indicate that the K2A -KO mice are more vulnerable to stressors, such as the novel environment. This would not be surprising, considering the importance of NE and other catecholamines, and their receptors, in regulating the CNS responses to acute stress (Arnsten, 1998). The reduction in locomotor activity in the open arena assessment, carried out as part of the primary behavioral screen (Table 1B), does not seem to denote any general hypoactivity of the K2A -KO mice, since no genotype di¡erence in total activity scores was detected during the ¢rst 5 min of the open ¢eld test (results not shown). The increased frequency of Straub tail in K2A -KO mice, observed after transfer to the open arena, may also be linked to the altered stress reactivity of the mutant mice, since both

stress and the administration of imipramine, an antidepressant drug inhibiting NE uptake, have been shown to induce Straub tail reaction in rodents (Katz, 1979; Molina et al., 1990). Other di¡erences detected in the primary behavioral screen included impairment in contact righting re£ex performance as well as in the rotarod test, i.e. in measures of motor coordination. The slightly inferior motor coordination skills of K2A -KO mice, observed in tests performed in non-trained animals, may also have been a consequence of the increased stress caused by the novel test environment. However, one cannot rule out the possibility that the lack of the K2A -AR would lead to an alteration in the control of muscle tone, since K2 -AR are known to modulate spinal re£exes (Clarke et al., 2001). For example, tizanidine, an K2 -AR agonist, is clinically used in the treatment of spasticity in patients with upper motor neuron dysfunction (Kita and Goodkin, 2000). No symptoms of spasticity were, however, evident in the tests applied to address this issue. Since the results from the primary behavioral screen indicated that the phenotype of the K2A -KO mice was associated with alterations in anxiety-related behaviors, the behavioral responses of the mice were further analyzed in tests measuring anxiety. We observed that the K2A -KO mice had increased anxiety, evidenced by a reduced number of exploratory head dips and a reduction in the time spent in the open arms of the elevatedplus maze. A decrease in rearing behavior of the K2A -KO mice in the open ¢eld test also suggests an anxiogenic phenotype of the KO animals. In a recent study with rats, administration of an antisense oligodeoxynucleotide against K2A -AR into the region of locus ceruleus caused an anxiolytic-like e¡ects in the elevated-plus maze test (Shishkina et al., 2001). However, opposite e¡ects of lifelong absence of the K2A -AR and locally administered antisense against the receptor are not entirely unexpected, considering the extensive expression of the receptor in the CNS (Scheinin et al., 1994). Anxiolytic drugs have been proposed to function in the marble burying test, a mouse model for detecting compounds with antianxiety properties, by decreasing the number of marbles buried (Broekkamp et al., 1986). However, the validity of the marble burying test as an isomorphic model of anxiety is not clear (Njung’e and Handley, 1991). In light of the expected response to anxiolytic drugs, the markedly reduced number of marbles buried by K2A -KO mice might re£ect an anxiolytic e¡ect of the mutation. All other measures for assessing anxiety-related behaviors in the current study argue against this interpretation. In addition, increased anxiety in K2A -KO mice was recently observed using another mouse model of anxiety, the light^dark paradigm (Schramm et al., 2001). In their paper, Schramm and co-workers reported increased anxiety of K2A -KO mice only after injection stress, whereas when no injections were given, there were no di¡erences between the K2A KO and WT mice. However, the currently observed genotype di¡erence in the marble burying test may be totally unrelated to the level of anxiety of the animals. The interpretation that rodents have less anxiety when

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they are burying foreign objects is based on the assumption that the animals would consider marbles startling by virtue of their novelty. Alternatively, it has been suggested that the burying behavior would be rewarding or that it is compulsive (Broekkamp et al., 1986). Nevertheless, the noradrenergic system and K2 -AR have recently been shown to participate in regulating the responses measured in the marble burying test, as evidenced by decreased marble burying behavior after administration of K2 -agonists (Millan et al., 2000) and after drugs inhibiting NE uptake (Millan et al., 2001). To investigate the validity of the marble burying test as a possible model for detecting anxiety, it would be interesting to evaluate the performance of other genetically modi¢ed mouse strains reported to have phenotypes with increased anxiety, such as mice lacking the gene for preproenkephalin, the 5-HT1A receptor, or the dopamine D4 receptor (Ko«nig et al., 1996; Ramboz et al., 1998; Dulawa et al., 1999). Interesting di¡erences in the diurnal patterns of activity were detected in the 24-h locomotor activity measurement. The K2A -KO mice exhibited clearly blunted increases in activity during the dark phase. However, there was no di¡erence between the K2A -KO and WT mice during the light phase. A possible explanation for the abnormal circadian rhythm of the K2A -KO mice could be their altered sympathetic regulation due to loss of K2A -AR-mediated inhibition of sympathetic tone, leading to altered sympathetic control of melatonin synthesis. By stimulating the production of melatonin in the pineal gland, NE is known to be an important part of the circuitry controlling the diurnal activity pattern of mammals (Sugden, 1989). Activation of central K2 -adrenergic receptors with K2 -AR agonists, such as medetomidine, also modulates pineal melatonin production through inhibition of the synthesis of this hormone (Mustanoja et al., 2001). The loss of K2A -AR resulted in increased levels of MHPG, the main metabolite of NE, in all brain regions studied. NE turnover, calculated as a ratio of MHPG to NE, was also consistently increased throughout the brain. These results were expected, considering the role of presynaptic K2A -AR as the principal inhibitory autoreceptor regulating catecholamine release (Altman et al., 1999), although K2C -AR are also required for normal presynaptic control of transmitter release (Hein et al., 1999; Trendelenburg et al., 1999, 2001). Also in accordance with the current results, an increase in NE turn-

297

over has previously been shown in the hippocampus of mice with dysfunctional K2A -AR protein (Lakhlani et al., 1997). Lack of signi¢cant di¡erences in the turnover of other monoamine neurotransmitters does not provide support for the notion of K2A -AR as an K2 -AR subtype with marked presynaptic heteroreceptor functions, but this needs to be further investigated using appropriate stimulation paradigms. Indeed, recent studies on mice lacking the K2A -AR, K2B -AR, K2C -AR or both K2A /K2C AR indicate that in hippocampal and cortical serotonergic nerve terminals, both K2A - and K2C -AR account for the K2 -mediated inhibition of 5-HT release (Scheibner et al., 2001). Interestingly, our previous work using mice with altered K2C -AR expression indicated a modulatory e¡ect of the K2C -AR subtype on in vivo turnover of DA and 5-HT, but not NE (Sallinen et al., 1999). The present study highlights the importance of a single adrenergic receptor subtype in multiple aspects of behavior and physiological responses in mice. The results not only con¢rm the anxiogenic phenotype of the mice with targeted inactivation of the K2A -AR gene, previously reported by another group (Schramm et al., 2001), but also reveal the signi¢cance of the K2A -AR subtype in the control of the diurnal pattern of locomotor activity. The results also emphasize the consequences of the K2A -KO mutation on the sensorimotor performance of the animals, expressed here most clearly as impairment in motor coordination and alterations in autonomic functions, and also as a decrease in the locomotor response to a novel environment. The unexpected decrease in aggressive behavior observed in the K2A -KO mice may re£ect an exaggerated reaction to stress, rather than an actual decrease in the emotional aggressive state of the mice. Many of the observed di¡erences possibly result from the increased NE turnover and synaptic concentrations both in the CNS and in the periphery of K2A -KO mice. In conclusion, the study provides detailed primary behavioral characteristics of the mice lacking functional K2A -AR protein and generates additional hypotheses for further studies of this physiologically important receptor.

Acknowledgements,Dr. Jouni Sirvio« is gratefully acknowledged for critical comments, and Dr. John Altman for generation of the K2A -KO mice. Pa«ivi Saikkonen is acknowledged for skilful technical assistance. The work was supported by The Technology Development Center of Finland (TEKES) and Orion Corporation, and by grants from the Pharmacal Research Foundation, Finland and the H. Lundbeck Foundation, Finland.

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