Motor and associative deficits in D2 dopamine receptor knockout mice

Int. J. Devl Neuroscience 20 (2002) 309–321

Motor and associative deficits in D2 dopamine receptor knockout mice S.C. Fowler a,b,c,∗ , T.J. Zarcone b , E. Vorontsova b , R. Chen a a

Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66045, USA Schiefelbusch Institute for Life Span Studies, University of Kansas, Lawrence, KS 66045, USA Department of Human Development, 4011 Dole Building, University of Kansas, Lawrence, KS 66045, USA b

c

Received 28 November 2001; received in revised form 4 February 2002; accepted 18 February 2002

Abstract Behavioral abnormalities produced by D2 dopamine receptor gene deletion in mice have been attributed either to resulting Parkinson-like features (i.e. response slowing and response initiation difficulties) or to behavioral deficits contributed by alleles of the originating 129Sv strain. Three strategies were used to address these conflicting hypotheses: (1) we used mice congenic at n10 backcross into the C57BL/6 line to minimize the 129Sv contribution; (2) we compared mice that were wild-type (+/+), heterozygous (+/−), or homozygous (−/−) for the D2 gene with the two most relevant inbred lines (129Sv and C57BL/6) and (3) we used both conventional and novel behavioral assessment methods. Behavioral attributes were expressed in terms of locomotor activity, wall rearing, rotarod performance, operant response acquisition, operant response performance, lick dynamics (force, rhythm), grip strength, and tremor in response to harmaline challenge. Results showed that, compared to controls, the −/− mice exhibited longer duration wall rears, retarded operant response acquisition, increased latencies to move from the operandum to the reward well, and exaggerated response to harmaline. Age was investigated as a variable (10–11 weeks versus 41–44 weeks of age) in the locomotor activity and wall rear assessments. A gene dosage effect (deficits in the +/− mice) on these two variables became apparent in the older mice. Taken together, the results showed that mice without the D2 gene exhibited Parkinson-like behavioral features that were not easily attributed to alleles contributed by the 129Sv strain, but were consistent with basal ganglia dysfunction. © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. Keywords: D2 dopamine receptor; Gene dosage effect; Harmaline challenge

1. Introduction Brain dopamine systems play a prominent role in our current understandings of Parkinson disease (Hornykiewicz, 1972), schizophrenia (Carlsson, 1988), habit-based learning (Mishkin et al., 1984; Packard, 1999; Hollerman et al., 2000), repetitive behaviors (Canales and Graybiel, 2000) and reward processes (Schultz, 1998). Advances in the experimental analyses of these systems as they relate to behavior in the whole mammalian organism have been accelerated by the develop of transgenic mice with targeted mutation for selected components of these dopamine systems (e.g. Xu et al., 1994; Giros et al., 1996). In particular, the targeted deletion of the D2 receptor (Baik et al., 1995) resulted in a phenotype having a marked hypoactivity trait accompanied by the expected lack of response to the D2-like receptor antagonist, haloperidol. Baik et al. (1995) proposed that their D2 knockout mouse displayed Parkinson like features. ∗

Corresponding author. Tel.: +1-785-864-0715; fax: +1-785-864-5202. E-mail address: [email protected] (S.C. Fowler).

A subsequent paper by Kelly et al. (1998) questioned this hypothesis and instead emphasized the importance of both background strain and gene dosage in producing the motor abnormalities in D2 knockout mice. Kelly et al. (1998) and earlier papers (e.g. Gerlai, 1996) raised awareness of the importance of the background strain in governing the phenotypic effects of targeted gene deletion. In the initial experiments on D2 −/− mice, the influence of alleles from the embryonic stem cell line (129Sv) was potentially large given the limited number of backcrossings to the host strain (C57BL/6). One of the purposes of the present work was to repeat some of the earlier behavioral observations made on D2 −/− mice (Baik et al., 1995; Kelly et al., 1998) but to do so with mice in the n10 generation of backcross into the C57BL/6 strain in which the probability of expression of 129Sv alleles linked to the D2 pseudogene is very low. A second objective of the present work was to introduce new methods of behavioral assessment that emphasize the measurement of the force and/or duration of specific motor acts (e.g. grip strength, wall rearing, latency to move from operandum to reward hopper, force and rhythm of tongue

0736-5748/02/$22.00 © 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 7 3 6 - 5 7 4 8 ( 0 2 ) 0 0 0 0 9 - 6

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movements during licking). Precise measurement of specific acts might make it possible to identify Parkinson-like motor phenomena such as reaction time slowing (e.g. Muller et al., 2000) and/or bradykinesia (e.g. Obeso et al., 2000). In addition, locomotor activity and rotarod procedures commonly used in the study of transgenic mice (Crawley et al., 1997) were also used to provide the opportunity to compare the n10 D2 −/− mice with the earlier results on D2 −/− mice obtained with these methods (Baik et al., 1995; Kelly et al., 1998). Although the D2 knockout mice used in this study were a congenic strain (Silver, 1995, defines congenic as n10 or more generations of backcross), mice from established 129Sv and C57BL/6 inbred strains should be studied for comparison purposes. The inbred C3H/He mouse was also included for comparison to serve as a “sensory deficit control” to assess any effect that reduced visual function may have on the behaviors that were measured. The C3H inbred mouse is known to undergo retinal degeneration (Schnitzer and Beane, 1976). Although it has not been reported for D2 −/− mice, it is possible that the lack of D2 receptors would have visual effects because D2 receptors are expressed in retina (e.g. Cellerino et al., 1998). Because D2 receptor density declines with age (in humans: Wong et al., 1997, in mice: Leprohon-Greenwood and Cinader, 1987), we conducted the locomotor activity assessment first when the mice were 10–11-week-old and then again when they were 41–44-week-old. It was hypothesized that changes in phenotype with age would be most apparent in the +/− mice because they began life with a deficient number of receptors. Finally, harmaline, a tremorgenic agent (Llinas and Volkind, 1973), was used as a motor challenge in an attempt to unmask differences among +/+, +/−, −/− mice. Previous reports suggested that harmaline may have direct stimulatory effects on the basal ganglia (Batini et al., 1981) and is capable of intensifying tremor in lesion models of Parkinson’s disease (Lamarre et al., 1975). Because haramline, at relatively high doses, damages cerebellar Purkinje cells (O’Hearn and Molliver, 1993), the harmaline tremor measurements were conducted after all other tests were completed.

2. Experimental procedures 2.1. Subjects All mice used in the studies reported here were purchased from Jackson Laboratories (Bar Harbor, Maine). The D2 knockout mice (Drd2 (tm1Low); stock no. 003190) were supplied as six males 6–9 weeks of age upon arrival in the University of Kansas Animal Care Unit. The +/+ and +/− controls were also supplied in the same manner as six individual males each. The mice were the 10th generation of backcross into the C57BL/6 background strain (congenic n10), and they came in three different batches, arriving at three different times several weeks apart. Lack of simultaneous arrival complicated the schedule of behavioral assessments, and resulted in some mice being assessed at different ages. Table 1 shows the ages for each of the types of mice studied. The +/+, +/−, and −/− mice were genotyped for verification of genetic status in the laboratory of Robert A. White. The three inbred strains used for comparison purposes, C3H (stock no. 000659), 129Sv (stock no. 000691), and C57BL/6 (stock no. 000664), were supplied as six males each, 6 weeks of age upon arrival. In the individual home cages water was always available and food was provided (Harlan Teklad: NIH-31 mouse/rat sterilizable diet 7017). Animals were kept on a restricted feeding schedule for at least 10 days prior to the lick–force–rhythm and operant learning tests. The mice received 1–2 g of food 30 min after training sessions and body weight was monitored daily to ensure maintenance of constant weight. The amount of food was adjusted individually for each mouse to take account of potential individual differences and to compensate for conditions where substantial caloric intake occurred (e.g. drinking milk in a training session) during behavioral assessments. The milk used as the reinforcer was two parts water and one part sweetened condensed milk; Borden Foods Corp., Columbus, OH, USA. Mice were run in behavioral tests during the light portion of the light–dark cycle in the vivarium (lights on from 6:00 a.m. to 6:00 p.m.). Use of the animals was approved by the University of Kansas IACUC, and procedures adhered to the NIH Guide for the Care and Use of Laboratory Animals.

Table 1 Mean (and S.E.M.) age in weeks when each procedure began for each type of mouse Procedure

Type of mouse +/+

Activity and wall rears Rotarod Operant acquisition Lick–force–rhythm Operant performance Grip strength Activity and wall rears, and harmaline tremor

11.0 10.0 14.0 20.0 24.0 33.0 41.0

+/− (0) (0) (0) (0) (0) (0) (0)

9.8 8.8 18.7 23.3 27.3 36.3 44.3

−/− (0.4) (0.4) (1.5) (1.1) (1.1) (1.1) (1.1)

10.7 9.7 19.2 23.2 27.3 36.3 44.3

(0.3) (.3) (1.8) (1.8) (1.7) (1.7) (1.7)

129Sv

C3H

10.0 9.0 12.0 16.0 17.0 26.0 34.0

10.0 9.0 12.0 16.0 17.0 26.0 34.0

(0) (0) (0) (0) (0) (0) (0)

Each group contained five or six mice. The +/+ mice were congenic at 10 generations on the C57BL/6J line of inbred mice.

C57BL/6 (0) (0) (0) (0) (0) (0) (0)

10.0 9.0 12.0 16.0 17.0 26.0 34.0

(0) (0) (0) (0) (0) (0) (0)

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2.2. Instruments and procedures 2.2.1. Locomotor activity and wall rearing Behavior was recorded with a new type of measurement instrument that used a force plate and computer software to measure a wide range of behaviors derived from the subject’s force variations as it moved on a 28 cm square sensing surface (detailed description in Fowler et al., 2001a,b). In the present study, the focal measurements were distance traveled and tremor. Recordings were taken at 50 samples/s from four concurrently operating chambers serviced by a LabMaster interface and a PC computer. The force plate in each chamber was supported by four Model 31 Sensotec force transducers (0–250 g range). The force plate itself was made from aluminum honeycomb as previously described (Wang and Fowler, 2001). Two equations drawn from physics were used to compute the coordinate (x, y) position of the subject based on the force measurements at each of the four corners of the force plate: x = (X1 f1 + X2 f2 + X3 f3 + X4 f4 )/(f1 + f2 +f3 +f4 ) and y = (Y1 f1 +Y2 f2 +Y3 f3 +Y4 f4 )/(f1 +f2 + f3 + f4 ), where lower case represents variables, and upper case represents fixed constants. Upper case X and Y values are the positions of the four force transducers mechanically coupled to the force plate (e.g. X1 = 140 mm, X2 = −140 mm, X3 = −140 mm, and X4 = 140 mm). In this coordinate system the center of the plate is x = 0, y = 0. The f ’s symbolize the force measurements from each of the four force transducers. The distance between successive center of force coordinates was calculated from the distance formula: d = ((x1 − x2 )2 − (y1 − y2 )2 )1/2 , where d is distance in mm, x1 and y1 are the center of force coordinates at time 1, and x2 and y2 give the center of force coordinates at time 2. Distance traveled was simply the line integral of the successive positions in time. Tremor was estimated from the sum of the forces from the four transducers; this was referred to as the Fz force because it represents the vertical force vector (both positive and negative directions) perpendicular to the force plate. Analyses not reported here indicated that using Fz to estimate the tremor produced results no different from those obtained by calculating the tremor for each transducer separately and then combining the results. The Fz was used because it reduced the computational effort by at least a factor of 4. Calibration procedures are described in the cited methods paper (Fowler et al., 2001a). All mice were placed for 30 min in the actometer for three consecutive days of recording. Data were partitioned into six 5-min blocks so that potential differences in rate of habituation could be observed if present. Wall rearing is the term we have given to the mouse’s behavior when it rears up on its hind limbs and contacts the wall of the enclosure that confines an animal to the force plate. When a mouse contacts the wall, a portion of its body weight is off-loaded from the force plate to the wall. With properly tuned software, these events can be detected as transient negativities in the Fz(t) force record. In heretofore unpublished observations we developed a wall rear detection algorithm

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and validated it by comparing computer-scored rears with counts of rears obtained from video taped records of the animal’s behavior. For example, for one BALB/c mouse, the Pearson correlation between computer identified wall rears per 1 min bin and visually detected wall rears per 1 min bin was 0.842. For one C57BL/6 mouse, observed in the same manner, the correlation was 0.749. A negativity in Fz(t) of 3.3 g was required to define the beginning of a wall rear, and negativity of 0.6 g was required to define the end of the event. In addition, a negativity had to last a minimum of 0.2 s to qualify as a wall rear. The algorithm extracted the wall rear events from the first 30 min session for each mouse. The dependent measure was the median duration of these rears for each mouse. The duration of the rear was emphasized because it was thought to reflect any bradykinesia during the execution of these discrete responses. 2.2.2. Rotarod The apparatus was Accuscan’s variable speed SmartRod, which was computer controlled with software supplied by the company. The diameter of the rod was 3.0 cm. The test procedure was performed across four consecutive days, during which a mouse was placed on the rod rotating at 4 rpm for up to 120 s for one to three trials. If a mouse fell before the 120 s had elapsed, it received a second or third trial as needed to attain 120 s on the rod. The dependent variable was the longest time (latency to fall) on the rod for a given day. This procedure is identical to the procedure reported by Kelly et al. (1998), except that the best score was used, not zero, if a mouse failed to attain 120 s on any of the three trials. The 4 rpm speed was relatively low, but this speed was chosen on the basis of our experience indicating that some inbred strains have difficulty even at this low level of motor coordination challenge. 2.2.3. Operant disk press acquisition and performance Eight operant chambers, each enclosed in a separate sound-attenuating box with exhaust fan, were customized to measure disk pressing by mice (for details, see Fowler et al., 2001b and Zarcone and Fowler, 2001). An intelligence panel was mounted on the front of each mouse chamber (23.5 cm × 21.5 cm × 18.5 cm). The house light at the top of the panel contained a white 24 V bulb (GE 1219) mounted behind a translucent Plexiglas cover. A sonalert speaker was connected to the houselight and mounted to the right of the houselight behind the intelligence panel. The sonalert generated a 70 dB tone whenever the houselight was turned on. The “reward hopper” was located below and to the right of the house light. A photo beam detected entries into and withdrawals from the reward hopper. An electromechanical dipper (Gerbrands, G5600 GS–RH) mounted just behind the reward hopper and outside the chamber presented 0.05 ml of sweetened-condensed milk for 5 s in the reward hopper via a hole located 3 cm deep into the hopper. A baffle (an L-shaped piece of aluminum that blocked the top half of the hopper) was added to prevent the animal from inserting

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more than the front half of its body into the hopper. A 0.5 cm hole was located on the intelligence panel 15 cm below the house light and 0.5 cm above the cage floor. A force-sensitive disk (based on a Model 31 Sensotec (Columbus, OH) load cell with a 0–250 g range) was positioned 0.2 cm behind the hole. A LabMaster (Scientific Solutions, Mentor, OH) interface and other custom built interfacing connected the experimental chambers to a 486 DX2 PC computer and controlled the house light, sonalert and dipper while collecting data from the force-transducer operandum and hopper photodetectors. The force–time waveform data produced by presses to the disk were measured with a computer controlled analog-to-digital converter, recording at 100 samples/s, thereby permitting the recording of number of disk press responses. Software also allowed for the measurement of the time interval between disk presses and entry into the hopper. The software controlling these chambers was written by T.J. Zarcone and S.C. Fowler. Prior to operant disk-press training, all mice received access to sweetened condensed milk (one part Borden’s Sweetened Condensed milk plus two parts tap water) in seven daily 20 min sessions of dipper presentation (5 s available alternating with 1 s unavailable). This was followed by nine sessions of a response-priming procedure in which milk was smeared onto the disk with a milk-saturated cotton swab and forces on the disk of 1.0 g or more produced dipper presentation for 5 s. Disk presses that followed a hopper entry within 5 s (i.e. the disk-hopper rate) was taken as the measure of learning of the disk-press–food-delivery relationship. Disk-hopper rate is a better measure of learning than disk-press rate because the disk-hopper rate was not influenced by disk presses that occurred during the course of consuming the bait (which was on the operandum) early in the session. Following the operant acquisition phase by an interval of 5–8 weeks (depending on the batch in which the mice arrived in the laboratory) mice received 23 consecutive days of operant training (no baiting, but disk presses produced milk reward—the “performance” phase). The last five of these sessions were averaged for each mouse for purposes of statistical analysis of the performance phase. The disk-hopper rate (as in the acquisition phase) and the latency from the end of a disk press to entry into the hopper served as the two dependent variables. The latter variable was selected for study on the basis of the report (Liao et al., 1997) that Parkinson-like slowing of response after 6-OHDA treatment was observed with this variable measured in rats. Recording irregularities (sticky transducers, resulting in occasional extinction contingency for several subjects) were experienced during the acquisition phase of training of the 129Sv, C3H, and C57BL/6 mice, and data for these mice are not presented. 2.2.4. Lick–force–rhythm assessment The apparatus has been described in other publications (Fowler and Mortell, 1992; Fowler and Wang, 1998; Wang and Fowler, 1999). Two simultaneously operating lick-measurement chambers were used. Each chamber had

a front panel containing a 6 cm square hole that gave access to a 6 cm cubic transparent plastic enclosure with a 12 mm diameter circular hole on its lower horizontal surface (at cage floor level). The mouse extended its tongue down through this hole to reach the force-sensing operandum. The operandum was an 18 mm diameter aluminum lick disk centered 2 mm beneath the circular hole in the plastic enclosure and rigidly attached to the shaft of a Model 31 load cell (Sensotec, Columbus, OH, USA). Sweetened milk could be pumped into the side of the disk and up through the center of the disk through a 0.56 mm hole drilled into the lick disk. A peristaltic pump (series E at 14 rpm; Manostat Corp., New York, NY, USA) was fitted with a solid-state relay (Digikey, Thief River Falls, MN, USA) and was controlled by computer interfacing (LabMaster, Solon, OH, USA) to deliver 0.015 ml milk when activated for 0.20 s. The transducer was mounted on a vertical micrometer shaft that allowed precise control of the lick distance from the inside of the plastic enclosure to the top surface of the operandum. A 386-PC computer recorded in real time the transducer’s force–time output sampled at 100 samples/s. The transducer gain was calibrated to resolve force to 0.2 g. The force threshold for lick detection was 1.0 g, and the force criterion required to advance the count for milk delivery was also 1.0 g. Illumination in the lick chamber was provided by a single GE 1819, 24 V DC light bulb located on the top center of the left side panel. Custom software was written by S.C. Fowler. After gradual adaptation to the restricted feeding regimen, mice were individually introduced to the lick chamber for daily 2 min sessions. All sessions began with automatic pump activation and delivery of 0.015 ml milk onto the lick disk (priming pulse). Ten licks were required for the delivery of each subsequent pulse of 0.015 ml milk onto the lick disk. Mice were given the opportunity to lick milk from the disk for 10 consecutive days. Data from the last three of these days were averaged to yield three measures for each mouse: number of licks in 2 min, mean peak force, and lick rhythm (the reciprocal of the lick period for licks within bursts). 2.2.5. Grip-strength assessment The apparatus was a horizontally mounted Model 31 force transducer (250 g range, Sensotec) interfaced with a PC computer via a Labmaster Interface (Scientific Solutions). In the test, the mouse’s paws gripped a wire bail of triangular shape. The base of the triangle gripped by the mouse was the 2 cm-wide base of the triangle, and the apex of this triangle was attached to the force transducer sensing shaft. The customized data acquisition software, written by S.C. Fowler, sampled the force signal at 100 samples/s. The grip-strength test was originally developed by Tilson and Cabe (1978) for use with rats. To perform the test, the examiner grasped a mouse by the base of the tail while its front paws gripped the wire bail attached to the force transducer. The examiner kept a mouse’s torso horizontal to the floor and allowed its forelimbs to flex. A mouse was held in this position for

S.C. Fowler et al. / Int. J. Devl Neuroscience 20 (2002) 309–321

1–2 s by the examiner, and then, over an approximately 2 s period, the examiner continuously increased his/her pull force on the mouse’s tail until the mouse released the bail. The peak force of each trial was the grip strength for that trial. Three trials were performed about 20 s apart for each mouse, and the maximum peak force across the three trials was used for statistical analysis. The procedure was conducted on two successive days, and the dependent variable used for analysis was the average of the results across the 2 days. 2.2.6. Harmaline tremor and locomotor activity Harmaline hydrochloride (Sigma–Aldrich) was dissolved in saline and injected i.p. immediately before a 30 min session in the force plate actometer. All mice received 15.0 mg/kg (as the salt) in a volume of 5 ml/kg. On the day before the harmaline treatment, mice were run for one 30 min session immediately after a saline injection. This saline session afforded an opportunity to observe the effect of aging on the D2 knockouts and controls which had their initial actometer session 8 months prior to this re-testing. Tremor was measured as previously described (Fowler et al., 2001a). Each 30 min Fz(t) force recording was partitioned into 180 successive time series of 10.24 s. Then 180 fast Fourier transforms were performed. The resulting 180 power spectra (with a frequency range from 0 to 25 Hz) were saved, and the integrated power in the 10–15 Hz frequency band for each power spectrum was computed and summed across the 180 power spectra to yield a single quantity for each mouse for each session. This measurement of tremor was reflective of both the duration and intensity of the tremor induced by harmaline during the 30 min recording session. 2.2.7. Statistical methods Analysis of variance (ANOVA) and Tukey HSD post hoc tests were used to evaluate the data. Effects were considered significant when P < 0.05. Computations were performed in SYSTAT (SPSS Science, Chicago, IL). In instances of heteroscedasticity, logarithmic transformations were performed before the ANOVA computations were undertaken. Rotarod latency to fall, operant disk-hopper rates in acquisition, and grip peak forces were the variables that were logarithmically transformed. 3. Results 3.1. Locomotor activity Fig. 1 shows distance traveled as a function of day (1–3) and as a function of time (1–6) within each session for each of the six types of mice. A three-way repeated measures ANOVA (type of mouse × day × block within day) applied to these data yielded a significant effect of type of mouse, F(5, 26) = 12.153, P < 0.001, a significant day effect, F(2, 52) = 42.775, P < 0.001, and a significant

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within-session effect, F(5, 130) = 158.345, P < 0.001. The main effects of type of mouse are shown in Fig. 2. By Tukey HSD post hoc test, the −/− mice were significantly lower in activity than the +/+ and +/− mice, which did not differ significantly from each other. The 129Sv strain was significantly lower in activity than all the other mice, except for the −/− mice. Despite the graphic separation in Fig. 2, the 129SV mice were not significantly lower than the −/− mice as determined by pair-wise post hoc comparison. Evidence for a modulating effect of type of mouse on the shape of the within-session habituation function can be obtained from the type-by-time-block interaction. In the overall ANOVA, this interaction was significant: F(25, 130) = 3.069, P < 0.001. To further specify the nature of this interaction, separate post hoc ANOVAs were performed on the +/+, +/−, −/− mice and on the three inbreds. No interaction of type-by-time-block was detected for the knockouts and controls (i.e. +/+, +/−, and −/− comprised type in the ANOVA). However, when the inbred strains were taken together, type- and time-block interacted significantly, F(10, 75) = 5.329, P < 0.001. This latter result confirms the different within session slopes (i.e. shapes of within session habituation functions; see Fig. 1, particularly day 3) for the inbred strains but not for the knockouts and their controls. To obtain a clearer account of the nature of the within-session trends for day 3 for the three inbred strains, trend tests were carried out for each strain separately. For the C3H mice the only significant trend was a strong linear one: F(1, 5) = 44.053, P = 0.001, whereas the strongest trend for the 129Sv mice was quadratic: F(1, 5) = 129.767, P < 0.001. In the case of the C57BL/6 mice the shallow habituation curve within session 3 was primarily linear, F(1, 5) = 14.720, P = 0.012, and the quadratic trend was not significant. In Fig. 3 (gray bars) are shown the locomotor activity data for one session when the knockout and controls were about 41–44-week-old. These data were taken from the saline control session which preceded the harmaline challenge, results for which are described below. A one-way ANOVA applied to the saline data indicated a significant effect of type of mouse, F(5, 27) = 14.496, P < 0.001. Post hoc tests showed that the +/− and the −/− mice were significantly lower than the +/+ mice. By the same method the 129Sv mice (34-week-old at this time) were not significantly different from the D2 knockouts. It should be noted that these data on the older mice revealed a gene dosage effect that was not present when the mice were tested at 9–11 weeks of age (compare Figs. 2 and 3 (gray bars) for the +/− mice). 3.2. Wall rears The wall rear algorithm was applied to the data from each mouse’s first 30 min of exposure to the actometer (see Fig. 4). This session was selected because previous unpublished work in our laboratory suggested that wall rearing was prominent when spontaneous locomotor activity levels

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Fig. 1. Group mean locomotor activity expressed as distance traveled in 5 min blocks during 30 min sessions for the indicated types of mice (10–11-week-old). D2 knockout mice were comparatively hypoactive as were the 129Sv mice. Brackets indicate ±1 S.E.M. When brackets are not visible, they are smaller than the respective plot symbol. Analysis of variance statistics are provided in the text.

were relatively high, and the data in Fig. 1 showed that the first session of exposure resulted in the highest levels of locomotor activity. As shown in Fig. 4 the −/− mice exhibited wall rears with durations significantly longer than any other type of mouse (P < 0.05 for each comparison by Tukey HSD post hoc test). In contrast, the number of wall rears was not significantly affected by type of mouse. The wall rear analysis was conducted again for the +/+, +/−, and −/− mice only on the saline day that preceded the harmaline challenge. The ANOVA on these data was again significant, F(2, 12) = 5.172, P = 0.024, with the −/− mice having significantly longer duration rears than the +/+ mice. The +/− mice had scores that fell between the −/− and +/+ mice, suggesting the tendency for a gene dosage effect to emerge with advancing age. As in the initial testing, the number of wall rear events was not significantly affected

by genetic status in the second test when the mice were 41–44-week-old. 3.3. Rotarod Fig. 5 shows the latency-to-fall data for the six types of mice for the four successive days of rotarod testing. A two-way repeated measures ANOVA indicated a significant effect of type of mouse, F(5, 30) = 6.178, P < 0.001, a significant effect of day, F(3, 90) = 20.549, P < 0.001, and a significant day-by-type interaction, F(15, 90) = 2.999, P = 0.001. Inspection of Fig. 5 suggested that the interaction effect was the result of across-day improvement in performance of the −/− and 129Sv mice. Post hoc test were run after a simple main effects ANOVA (collapsing data across the 4 days), and it showed that the −/− mice were

S.C. Fowler et al. / Int. J. Devl Neuroscience 20 (2002) 309–321

Fig. 2. Locomotor activity expressed as the total distance traveled in the 3 days of testing for the indicated types of mice when they were 10–11-week-old. See text for analysis of variance results. Brackets as in Fig. 1. By post hoc comparison, −/− mice were significantly lower than +/+, +/− and C57BL/6 mice (∗ ); the 129Sv mice were significantly lower than all other types except the −/− D2 mice (# ).

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Fig. 4. Group means for the median wall rear duration for the first session of locomotor activity assessment shown in Fig. 1. Brackets as in Fig. 1. The −/− mice were significantly higher than any of the other types of mice (∗ ).

3.4. Lick dynamics significantly poorer in performance than all the other types except for the 129Sv inbred strain. On day 1 of the rotarod behavioral assessment, data in Fig. 5 (filled bars) suggest the presence of a gene dosage effect (i.e. the +/− mice had a mean performance that was located about halfway between the +/+ and −/− mice). However, this effect was only partially supported (+/+ compared to +/−: P = 0.10) by the Tukey post hoc test performed on the day 1 data only. More data will be needed to decide this question. It is clear from Fig. 5 that the +/− mice on days 2–4 were more similar in performance to the +/+ mice than they were to the −/−.

All mice learned to lick milk from the force-sensing disk, and data for lick mean peak force, lick rhythm, and number of licks in 2 min are shown in Fig. 6. A one-way ANOVA on the force data yielded a significant effect of type of mouse, F(5, 30) = 6.634, P < 0.001. Although the +/− mice had lower average peak forces than the +/+ and −/− mice, these three groups did not differ significantly from each other. The 129Sv strain was significantly lower in peak force than the +/+, −/−, and C57Bl/6 mice. The level of lick force for the mice with the C57BL/6 heritage observed here was

Fig. 3. Distance traveled in 30 min sessions after a saline injection and after harmaline treatments conducted when the +/+, +/−, and −/− mice were 41–44-week-old and the three inbred strains were 34-week-old. Brackets as in Fig. 1. The +/− and −/− mice were significantly below the +/+ controls on day 1 (∗ ).

Fig. 5. Group mean rotarod performance across four consecutive days of testing at speed of 4 rpm on an Accuscan SmartRod. Brackets as in Fig. 1. The horizontal bracket indicates that, for the 4 days combined, the −/− mice were significantly lower than all of the other types of mice, except for the 129Sv mice.

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Fig. 7. Group mean rates of successful disk-press–milk-reward responses over 9 days of the response priming procedure in the operant chamber. Brackets as in Fig. 1.

with the relatively low lick rhythms previously reported for the C57BL/6 strain compared to the BALB/c strain and the outbred CD-1 stock (Wang and Fowler, 1999). The one-way ANOVA on number of licks in 2 min produced a significant test statistic, F(5, 30) = 3.310, P = 0.017; however, the only pair-wise comparison that reached significance was the 129Sv strain being significantly higher than the +/− mice. Again, number of licks for the C57BL/6 or C57BL/6-derived mice were comparable to previously reported results (Wang and Fowler, 1999). 3.5. Operant behavior

Fig. 6. Results of the lick dynamic assessment for the D2 knockouts, controls and inbred strains. See text for analysis of variance statistics. Brackets as in Fig. 1. The 129Sv mice were significantly lower in peak lick force than +/+,−/−, and C57BL/6 mice (∗ ); both the 129Sv and C3H mice had significantly higher lick rhythms than +/+, +/−, −/−, and C57BL/6 mice (# ); the 129Sv mice were significantly higher than the +/− mice (& ).

consistent with previous measurements on C57BL/6 mice from Charles River laboratories (Wang and Fowler, 1999). In regard to lick rhythm, the F-test was significant, F(5, 30) = 12.564, P < 0.001, but the +/+, +/−, and −/− mice did not differ from one another by post hoc comparisons. The primary source of the significant F-test was the higher lick rhythms of the 129Sv and C3H mice compared to those mice with a C57BL/6 heritage. This result was consistent

3.5.1. Acquisition Fig. 7 presents the operant data for the +/+, +/−, and −/− mice for the acquisition of the disk-press–milkconsumption behavior. A two-way, repeated measures ANOVA (three types of mice by 9 days) resulted in a significant effect of type, F(2, 15) = 7.330, P = 0.006, a significant effect of day, F(8, 120) = 12.941, P < 0.001, and a significant interaction between type and day, F(16, 120) = 3.882, P < 0.001. A post hoc two-way ANOVA using only the +/+ and the +/− mice showed no differences. These results confirm the poor performance of the −/− mice shown in Fig. 7. 3.5.2. Performance Although the −/− mice were slow to learn the operant contingency, they eventually learned to disk-press and consume milk from the dipper. Because of apparatus problems that occurred after the initial 9 days of acquisition some mice did not receive milk reinforcement on a few days. These irregularities made it impossible to determine the number of sessions required to train the −/− mice to respond consistently in the operant chamber. After the problems were eliminated, all mice received 23 consecutive daily sessions of operant training (no response priming). Data from the last 5 days of this phase were averaged for each mouse to

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Fig. 9. Effects of 15.0 mg/kg harmaline on the induction of tremor in the 10–15 Hz frequency band of the power spectra for the Fz(t) force recording from a 30 min session in the force plate actometer. The +/+, +/−, and −/− mice were 41–44-week-old at the time of this procedure, while the inbred strains were 34-week-old. See text for explanation of the quantitative methods. Brackets as in Fig. 1. Significantly greater than the +/+ mice (∗ ).

3.7. Harmaline tremor

Fig. 8. Data for steady-state performance of operant disk-pressing after all six types of mice had learned the disk-press-reward contingency. Brackets as in Fig. 1. The −/− had significantly longer latencies than any of the other five types of mice (∗ ); the disk-hopper rate of the C3H mice was significantly higher than any of the other types of mice (# ).

yield the performance data presented in Fig. 8. A one-way ANOVA for the disk-hopper latency (upper axes in Fig. 8) resulted in a significant effect of type of mouse, F(5, 26) = 41.082, P < 0.001. By Tukey HSD, the −/− were significantly slower than the other five types of mice. In regard to the disk-hopper rate, the one way ANOVA was significant, F(5, 26) = 5.868, P = 0.001. The post hoc analyses detected no differences among the +/+, +/−, and −/− mice on this measure. The C3H strain was significantly higher than all other types, except the −/− mice. Although the 129Sv mice had a lower mean rate than any other strain, this result was not significant by the Tukey HSD pair-wise comparison method. 3.6. Grip strength The one way ANOVA did not detect any significant differences between the types of mice in grip force (data not presented graphically). The mean forces (and S.E.M.) for the +/+, +/−, −/−, 129Sv, C3H, and C57BL/6 mice were 141.6 (11.3), 141.2 (9.5), 154.9 (31.0), 110.3 (13.4), 109.4 (4.3), 114.7 (10.5), respectively (units are g).

Inspection of the Fz(t) time series for each animal of each type (data not shown) confirmed that the characteristic 12–14 Hz rhythmic oscillations occurred in all mice at least some of the time. The expected distinctive intermittent loss of harmaline-induced tremor was also observed in the records for all mice. As explained in the Section 2, the tremor results were expressed in terms of a dependent variable that combined the intensity of tremor and its duration of occurrence. Data are shown in Fig. 9. A one-way ANOVA on these data for type of mouse was significant, F(5, 27) = 4.456, P = 0.004. The Tukey pair-wise post hoc procedure indicated that both the +/− and −/− mice displayed significantly more tremor than the +/+ mice. The three inbred strains had data that were graphically higher than the +/+ and lower than the +/− and −/− mice, but none of the pair-wise comparisons was significant. Overall, these data show that, compared to the appropriate control (i.e. +/+ mice), both the +/− and −/− mice exhibited an exaggerated response to harmaline. 3.8. Empirical summary Table 2 provides a summary of the empirical results. The ordering of the types of mice is based on the ordinal rank of the group means, with values for the measures descending from left to right. The “greater than” symbol indicates statistical significance and the symbol for “approximately equal to” indicates lack of significance based on pair-wise comparisons even in the face of modest numerical differences between the means. In three instances in the table, the −/−

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Table 2 Empirical summary for the performance of +/+, +/−, −/− dopamine D2 receptor knockout mice and three comparison inbred strains of mice Type of task and measure

Ordering of group meansa

Locomotor activity (Figs. 1–3) Younger age Older age

C57BL/6 ≈ +/+ ≈ +/− >C3h > −/− ≈ 129Sv C3H ≈ +/+ ≈ C57BL/6 > +/− >129sv ≈ −/−

Wall rears (Fig. 4) Number (not shown) Duration

No differences −/−  129Sv ≈ C3H ≈ C57BL/6 ≈ +/− ≈ +/+

Rotarod (Fig. 5) Latency to fall

+/+ ≈ C57BL/6 ≈ C3H ≈ +/−  129Sv ≈ −/−

Lick dynamics (Fig. 6) Force Rhythm Number of licks

C57BL/6 ≈ +/+ ≈ −/− ≈ +/− ≈ C3H >129Sv 129Sv ≈ C3H > C57BL/6 ≈ +/+ ≈ +/− ≈ −/− 129Sv >C3H ≈ C57Bl/6 ≈ +/+ ≈ −/− ≈ +/−

Operant Acquisitionb (Fig. 7) Disk-hopper rate

+/+ ≈ +/−  −/−

Performance (Fig. 8) Disk-hopper latency Disk-hopper rate

−/−  C3H ≈ +/− ∼ C57BL/6 ≈ +/+ ≈ 129Sv C3H > −/− ≈ +/+ ≈ +/− ≈ C57BL/6 ≈ 129Sv

Grip strength Peak force (not shown)

−/− ≈ +/+ ≈ +/− ≈ C57BL/6 ≈ 129Sv ≈ C3H

Harmaline tremor (Fig. 9) Integrated power (10–15 Hz)

−/− ≈ +/− >C3H ≈ C57Bl/6 ≈ 129Sv ≈ +/+

The −/− mice are indicated in a bold font to enhance visual identification of their ordering among the comparison types. a The “>” indicates greater than, “” indicates much greater than, and “≈” indicates not different. b Data not available for the inbred strains.

mice are underlined (duration of wall rears, disk-hopper latency in operant performance, and integrated power for harmaline tremor). These are behavioral markers that distinguish the −/− mice from their conventional +/+ controls as well as from the 129Sv strain’s performance.

4. Discussion The locomotor activity data reported here for the −/− mice and controls generally agree with results reported by Baik et al. (1995) and by Kelly et al. (1998) except for the +/− mice. The cited authors reported distinctive hypoactivity for the −/− mice compared to the +/+ mice while the +/− fell in between these extremes. In our data for the mice tested at 10–11 weeks of age, there was no evidence for a gene dosage effect in that the +/− mice did not differ from the +/+ mice; however, we observed hypoactivity in the +/− mice when they were 41–44-week-old. Moreover, in the current study, the 129Sv mice were even less active than the −/− mice, a result similar to the data obtained by Kelly et al. (1998). The discrepancy between our data on the +/− mice locomotor activity results reported by the two investigative teams cited above was probably the result of the presence of many more 129Sv alleles in their +/− mice than in ours which were derived from the n10 congenic

strain. In addition, a report using mice at the n5 generation obtained results similar to ours such that with young mice the +/+ and +/− types were closely similar (see Fig. 8 in Zahniser et al., 2000). Within-session statistical analyses performed on the activity data when the mice were young (Fig. 1) suggested that habituation curves for the +/+, +/−, and −/− mice were not significantly different (no interaction between time block and type of mouse was detected by ANOVA), a result that raises the possibility that the D2 receptor gene may not be involved in habituation processes as measured in this paradigm. Nevertheless, habituation data may reward analysis efforts as illustrated by the data from the inbreds. As described in the Section 3.1, each of the three strains on day 3 had distinctly different within-session trends in locomotor habituation. Identification of the underlying gene/brain mechanisms for these differences must await further experimental work. In contrast to when the mice were 10–11 weeks of age, when the +/+, +/−, and −/− were 41-44-week-old, a gene dosage effect on locomotor activity was observed (see, the three left-most gray bars in Fig. 3). This observation is consistent with the finding that D2 receptor density declines with age (Leprohon-Greenwood and Cinader, 1987; Wong et al., 1997). Because the D2 +/− mice have fewer D2 receptors when young (demonstrated by Baik et al., 1995), age-related

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receptor decline would be expected to lower dopamine system function more rapidly in +/− than in +/+ mice, making them more similar to −/− mice as age advanced. We are unaware of any direct measurements on aged +/− mice to confirm of this hypothesis. Nevertheless, this result, plus the comparative hypoactivity of the −/− mice, supports the idea that the hypoactivity trait of the −/− mouse is Parkinson-like. With regard to duration of wall rears, the current data support ethogramic characterizations of D2 −/− mice reported by Clifford et al. (2000, 2001). D2 −/− mice exhibited changes in rearing topographies relative to controls (Clifford et al., 2001). Thus, the current finding of prolongation of the wall rear may be correlated with the reported alterations in rearing topography in the D2 −/− mice. On the first day of exposure to the rotarod test, the −/− mice performed significantly below the +/+ mice, with the +/− mice falling in between, similar to results obtained by Baik et al. (1995), who reported data for only one rotarod session. Baik et al. (1995) did not assess the behavior of the 129Sv strain, but in our study their performance was highly similar to that of the −/− mice. The data reported here for the −/−, +/−, +/+, 129Sv, and C57BL/6 were similar to rotarod data presented by Kelly et al. (1998, Fig. 3a and c). The only notable difference between our rotarod data and those of Kelly et al. (1998) was that their n5 −/− mice reached the same level of performance after 4 days as their +/− and +/+ mice, whereas our n10 −/− mice were substantially below their +/− and +/+ counterparts on day 4 of rotarod experience (see Fig. 5). This relatively small discrepancy may have been the result of their use of a larger diameter rod of 6 cm compared to our 3 cm diameter rod, thereby presenting a possibly lesser motor challenge in their rotarod assessment. Operant response acquisition was delayed in −/− mice compared to +/+ and +/− mice. This result is consistent with the hypothesis that the striatum, where the preponderance of brain D2 receptors are located, is a vital part of the circuitry responsible for successful habit learning (Mishkin et al., 1984; Packard, 1999; Hollerman et al., 2000) or procedural learning (Knowlton et al., 1996). The −/− mice eventually learned the disk-press–milk-reward contingency, but recording problems prevented us from being able to characterize the learning rate differences quantitatively in terms of trials to reach asymptote. After all mice had attained relatively stable operant responding, our results indicated a specific deficit in the −/− mice in movement-related aspects of the performance. Mice lacking the D2 receptor exhibited a longer latency between the end of the disk-press and entry into the milk hopper. This delay likely had two components: response initiation and movement time. Although the contribution of each component could not be assessed with the measurements taken in the current study, both response initiation deficits and movement time deficits are consistent with motor abnormalities of Parkinson disease. In a similar paradigm with rats, Liao et al. (1997) reported the

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lengthening of operant-response–hopper-entry latency as a consequence of selective neurotoxic damage to the nigrostriatal system. Using −/− mice and controls Risinger et al. (2000) reported operant response deficits associated with the missing D2 receptor. One of the most striking features of the results reported here for operant performance is the fact that the 129Sv strain did not show the increased disk-hopper latency seen for the −/− mice. That is, the performance of discrete learned acts revealed the −/− deficits as separate from the traits of the 129Sv strain that were similar to those of the −/− mice in the actometer and rotarod assessments. Harmaline challenge suggested that mice deficient in D2 receptors were more sensitive to this drug’s effects than the +/+ and inbred mice. Inspection of the tremor records (not shown) indicated that a major portion of the greater response by the −/− and +/− mice was the time spent in frank tremor compared to the other mice. For unknown reasons, harmaline-induced tremor, which is believed to be a result of selective over-excitation of inferior olive neurons which in turn over-stimulate the cerebellum (e.g. Llinas and Volkind, 1973), is not expressed continuously (see discussion in Wang and Fowler, 2001). In the latter paper, it was hypothesized that periods of tremor are associated with the mouse’s attempts to locomote because harmaline suppress locomotion as was observed in the present study (Fig. 3). Thus, the more enduring tremor of the −/− and +/− mice may be a reflection of a deficit in learning the relation between locomotion attempts and the intense shaking which movement produces. We propose that the tremoring is similar to a punishing stimulus. On the other hand, greater harmaline response in the D2 deficient mice may be an exacerbation of Parkinson-like motor abnormalities that are amplified by pharmacological motor challenge (cf., Lamarre et al., 1975). Data from the lick dynamics task did not reveal any significant differences attributable to the missing D2 receptors. However, the lick rhythm data showed that C57Bl/6-derived animals exhibited performances appropriate to their background strain. Moreover, this rhythm of about 7 Hz was significantly lower than that of the 129Sv and C3H strains. The lick-rhythm data support the idea that the +/+, +/−, and −/− mice at congenic n10 were not appreciably influenced by residual alleles from the 129Sv strain that impact lick dynamics. One reason for including the lick dynamics assessment in this biobehavioral analysis was the finding by Skitek et al. (1999) that lick rhythm was slowed in rats with a unilateral lesion of the nigrostriatal system (i.e. a rodent model of Parkinson disease). Thus, it was expected that the −/− mice would also exhibit slower lick rhythms than appropriate controls. Graphically, the data in Fig. 6 were in accord with this hypothesis, but the effect was not large enough to reach statistical significance. Given that the sample sizes were small (n = 6 per group) in the current experiments, it is possible that significant rhythm slowing in the −/−mice would emerge from work using larger numbers of subjects per group. Of course, it is also possible that

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the relatively low lick rhythm of the C57BL/6 strain relative to 129Sv, C3H, CD-1, and BALB/c mice (the latter two reported previously in Fowler and Wang, 1998) may present a “floor effect” below which it is difficult to depress the lick rhythm. While the grip-strength assessment did not reveal any differences among the six groups of mice examined, this result was important for interpretative purposes because it demonstrated that the +/− and −/− mice did not have generalized problems with muscle weakness or with short-duration neuromuscular function that could have otherwise complicated the interpretation of the observed response slowing seen for wall rears and operant-reward latencies. Overall, the results support the idea that the behavior of the D2 knockout mouse has features that resemble those of Parkinson disease, such as response slowing and stimulus-response associative deficits. Moreover, these functional problems in the mouse cannot be easily attributed to either the originating stem cell line or to the background strain. Methodologically, the data suggest that conventional measures, such as locomotor activity or rotarod-based assessments, may fall short of detecting meaningful behavioral effects occasioned by the deletion of genes expected to have pronounced effects on the production and control of behavior. Instead, methods which permit the precise measurement of temporal attributes of clearly defined discrete behaviors (i.e. behavioral responses that have a well defined beginning and end) may be required to characterize fully the consequences of gene deletion in mouse neurobiological models.

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