Regulation of hind-limb tone by adenosine A2A receptor in rats

Neuroscience 159 (2009) 1408 –1413

REGULATION OF HIND-LIMB TONE BY ADENOSINE A2A RECEPTOR IN RATS Y.-N. WU,a,b J.-J. J. CHEN,a L.-Q. ZHANGb AND B. I. HYLANDc*

1997; Mandhane et al., 1997), whereas antagonists increase motor activity (Hauber et al., 1998), suggesting that endogenous adenosine, acting through the A2A receptor may have an anti-kinetic role. This stands in contrast to the pro-kinetic role of endogenously released dopamine, acting at the co-localized D2 receptors, evidenced by the cataleptic actions of D2 receptor antagonists (Hauber and Munkle, 1997). The enrichment and reciprocal interaction of A2A and D2 receptors in the basal ganglia suggested that manipulation of the A2A receptor may offer a new approach to alleviating Parkinsonian symptoms (Richardson et al., 1997; Xu et al., 2005), and indeed, a selective antagonist of the adenosine A2A receptor is showing promise in clinical trials (LeWitt et al., 2008). In addition to hypokinesia, Parkinson’s disease is also characterized by limb rigidity, expressed in clinical assessment as increased resistance to passive movement of the limb (Berardelli et al., 1983). Changes in levels of rigidity are thought to be contributed to by increased long loop reflex and co-contraction responses to imposed movement (Marsden et al., 1973; Lee and Tatton, 1975; Tatton and Lee, 1975; Burke et al., 1977; Mortimer and Webster, 1979; Berardelli et al., 1983; Rothwell et al., 1983; Tatton et al., 1984; Lee, 1989; Meara and Cody, 1992), although alterations in passive properties of tissues may also play some role (Watts et al., 1986). Two separate components contribute to changes in resistance to imposed joint rotation: elasticity and viscosity. Elasticity, referred to as stiffness, reflects the spring-like property of joints, whereas viscosity is a measure of the ability of the joint to dampen imposed movement. Stiffness is not velocity dependent, whereas viscosity is, due to a contribution from activation of the stretch reflexes (Latash, 1993). Accordingly, increases in viscosity can be demonstrated in hemiparetic spasticity (Lee et al., 2002). Quantification of rigidity in Parkinson’s disease has confirmed elevation in stiffness (Caligiuri, 1994), as would be expected. However, although subjective clinical assessment focuses on nonvelocity dependence of Parkinsonian rigidity, changes in velocity-dependent components can be detected in Parkinson’s disease with quantitative methods (Rothwell et al., 1983; Gregoric et al., 1988; Gresty, 1989; Teravainen et al., 1989; Lee et al., 2002), consistent with a role for changes in short or long-loop reflex components in this disorder (Lee, 1989; Bergui et al., 1992). A similar mixed picture is seen when limb rigidity is quantified in animals with neuroleptic (dopamine D2 antagonist) induced catalepsy. Thus, rats treated with the neuroleptic haloperidol show increased total resistance to

a Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan b Sensory Motor Performance Program, Rehabilitation Institute of Chicago, Chicago, USA c Department of Physiology, School of Medical Sciences, University of Otago, Post Office Box 913, Dunedin, New Zealand

Abstract—Adenosine A2A receptor agonists produce a hypokinetic state (catalepsy) that is believed to reflect antagonistic interaction of A2A and dopamine D2 receptors in the basal ganglia. In addition to catalepsy, pharmacological blockade of D2 receptors produces rigidity. However there are conflicting data about the effect of A2A agonists on muscle tone, with some reports indicating an increase, while other data suggest that A2A catalepsy is dominated by muscle hypotonia. We investigated the effect on resistance to imposed movements of systemic cataleptic doses of the selective A2A agonist CGS21680 (5 mg/kg), and compared it with the effect of the D2 antagonist raclopride (5 mg/kg), in rats. Total resistance is made up of elastic and viscous components. The elastic component is velocity independent, and is referred to as “stiffness,” whereas viscosity, which dampens responses to imposed movements, is velocity dependent. Using a method for quantifying total joint resistance that enabled separate identification of stiffness and viscosity, we found that during catalepsy evoked by either drug there was a clear increase in joint rigidity. Both CGS21680 and raclopride significantly increased joint stiffness, the velocity independent component of rigidity that is most affected in Parkinsonism. In contrast, the effect of CGS21680 on the velocity-dependent viscosity component was less robust than for raclopride, and did not reach significance, possibly reflecting an interaction with sedative effects via extrastriatal receptors. The effect of CGS21680 and raclopride on joint stiffness is thus consistent with previous findings suggesting functional antagonism of A2A and D2 receptors in the basal ganglia. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: rigidity, Parkinsonism, catalepsy, neuroleptic, dopamine D2, CGS21680.

Adenosine A2A receptors are very highly and relatively selectively expressed in the basal ganglia, where they co-localize with dopamine D2 receptors on striatal medium spiny projection neurons that project to the globus pallidus, forming the indirect pathway (Rosin et al., 1998; Svenningsson et al., 1999). Administration of A2A agonists systemically, or locally into the basal ganglia produces a hypokinetic state (Ferre et al., 1991; Hauber and Munkle, *Corresponding author. Tel: ⫹64-3-479-7342. E-mail address: [email protected] (B. I. Hyland).

0306-4522/09 $ - see front matter © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.01.068

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joint movement (Kolasiewicz et al., 1987; Lorenc-Koci et al., 1996) along with increased long-latency stretch reflexes, suggesting that a velocity-dependent component may be operating (Lorenc-Koci et al., 1996). Recently, we confirmed using the more selective D2 antagonist raclopride, and a method capable of segregating the two acutely variable sub-components of rigidity, that increases in total resistance are contributed to by increases in both stiffness and viscosity (Wu et al., 2007). The hypothesis of a reciprocal relationship of A2A and D2 receptors in modulating movement control circuits of the basal ganglia thus predicts that A2A agonists should also produce such rigidity. In support of this, increased muscle tone and EMG activity has been reported following intrastriatal injection of the A2A agonist CGS21680 (Wardas et al., 1999). However, some uncertainty remains about the effect of A2A agonists on muscle tone. The one previous study which quantified limb resistance found increases in muscle tone for only one direction of movement (Wardas et al., 1999), whereas parkinsonian rigidity is uniform. Furthermore, another study, which did not quantify limb resistance, reported that animals treated with systemic administration of this drug are flaccid, not rigid, and it was suggested that A2A agonist-induced akinesia might reflect a muscular hypo- rather than hypertonia (Rimondini et al., 1997). In the present study we therefore re-examined the effect of systemic cataleptic doses of the selective A2A agonist CGS21680, using a method we recently described for quantifying both the stiffness and viscous components of joint resistance in small animals (Wu et al., 2007).

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EXPERIMENTAL PROCEDURES Animals and experimental procedures Experimental procedures were approved by the National Cheng Kung University Medical College Animal Use Committee. All procedures were performed according to the Society for Neuroscience Policy on the Use of Animals in Neuroscience Research, and all efforts were made to minimize suffering and numbers used. Eight male Wistar rats (300 – 400 g, obtained from the Laboratory Animal Center, National Cheng Kung University, Taiwan) were used in the current study. The animals were housed in pairs in a colony room maintained on a 12-h light/dark cycle, with dark onset at 20:00 h. All animals had continuous access to food and water throughout the experiments. Rats were familiarized with the restraint apparatus for 30 min, twice a day for 1 week before the experiment. The rats received the selective dopamine D2 receptor antagonist raclopride (Sigma, St. Louis, MO, USA), the selective adenosine A2A agonist CGS21680 (Sigma), or vehicle (normal saline) injection in three different sessions every other day in a randomized order. Raclopride was injected intraperitoneally in a volume of 1 ml/kg, at a dosage of 5 mg/kg (Hemsley and Crocker, 1999). CGS21680 was also administrated intraperitoneally, at a volume of 1 ml/kg and dosage of 5 mg/kg (Rimondini et al., 1997). In control experiments, rats were injected with equivalent volumes of vehicle alone. Druginduced akinesia was quantified using bar tests 15–20 min after raclopride, CGS21680 or vehicle injection. For this, both of the rat’s forepaws were put on a horizontal acrylic bar, positioned 9 cm above the floor (Fig. 1). We recorded the time from placing of forepaws to the first complete removal of both of them from the support bar. The maximal testing duration was set at 180 s. To ensure that all tests were performed with a consistent minimum level of akinesia, rats only proceeded to rigidity testing if they showed a bar time of at least 30 s.

Biomechanical measurement of rigidity To quantify rigidity, we used a miniature muscle tone assessment system that we have recently developed, and which is described

Fig. 1. Drug induced akinesia. Frames taken from movies of bar-test sessions following vehicle, CGS21680 (CGS) and raclopride (Raclo) administration. Numbers in frames show time from the first frame, in s. For the vehicle session, frames are 1 s apart, whereas for the drug test sessions the frames are 10 s apart.

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in detail in Wu et al. (2007). This system provides precise measurement in the rat of the relationship between reactive resistance and joint angle during sinusoidal flexion/extension movements, from which the two major biomechanical determinants of total resistance to joint movement, elastic stiffness (K) and viscosity (B), can be derived (Lee et al., 2004; Wu et al., 2004, 2007; Chen et al., 2005). In brief, rats were secured in a restrainer for the testing, after prior habituation. The assessment system is composed of two isolated balloons which are mounted on the dorsal and ventral surface of the foot and connected to differential pressure sensors (DP45, Validyne Engineering Corp., Northridge, CA, USA) to measure reactive resistance to imposed movement, and an optical angle sensor (S720 Miniature Joint Angle Shape Sensor, Measurand Inc., Fredericton, New Brunswick, Canada) mounted over the ankle joint, to record joint angle. The rat’s foot was manually moved back and forth in a reciprocal pseudo-sinusoid movement within a set of limiters. During the imposed (passive) movements the pressure difference between the two balloons and the joint angle data were sampled at 500 Hz via an analog-to-digital converter with 12-bit resolution (DAQPad-6020E, National Instruments, Austin, TX, USA) (see Fig. 2A) controlled by LabView (National Instruments). The data were displayed in real-time for monitoring and then stored in a laptop computer for further signal processing using Matlab (The MathWorks, Natick, MA, USA). Biomechanical measures were performed before and then every 20 min after injection, for a total of nine post-drug tests per experiment (3 h). During each biomechanical test, 15 successive cycles of passive 100° sinusoidal movements were applied at each of five frequencies (1/3, 1/2, 1, 3/2 and 2 Hz). Production of the different frequencies was achieved by pacing to a metronome. To maintain consistency, we monitored the ankle angle on a real-time display in the LabView program, and also checked the oscillation frequency from off-line frequency spectral analysis (fast Fourier transform, FFT) of the angular displacement data. Experiments were not further analyzed if the range of movement did not reach the set limits, or if the dominant oscillation frequency of the manual, paced displacement was not located at the prescribed frequency. Frequency analysis was used to determine the actual dominant frequency achieved by our paced manual system, over the data acquisition period, and showed that the achieved frequencies matched well with the paced values.

Data analysis Biomechanical measurement. We derived two measures of rigidity (Latash, 1993): joint stiffness (K) and viscosity (B, Fig. 2). Stiffness reflects the spring-like action of the joint, is not velocity dependent, and was derived from the slope of a linear regression calculated for the hysteresis loop formed by plotting joint angle against reactive resistance (Fig. 2B). Stiffness was evaluated from data obtained at a low oscillation frequency (1/3 Hz), and hence low angular velocity, which diminishes the contribution of the velocitydependent stretch reflex. Joint viscosity, which reflects the damping effect of the joint including reflex components, and is therefore velocity dependent, was estimated as detailed previously (Lee et al., 2004; Chen et al., 2005; Wu et al., 2007). In brief, the viscous component B␻ for each limb oscillation frequency ␻ was estimated from the phase lag between displacement and resistance (Fig. 2A), which causes hysteresis in the plot of the reactive resistance (T) versus joint angle (Fig 2B). Viscosity (B) was then defined as the slope of the regression line for B␻ against frequency (Fig. 2C). Statistical analysis. For analysis of bar test data, the time the rat remained with forepaws on the bar was rescaled using square root transformation. Times from 0 to 0.08 min were scored as 0, 0.09 – 0.35 min⫽1, 0.36 – 0.80 min⫽2, 0.81–1.42 min⫽3, 1.43–2.45⫽4, and ⱖ2.25 min⫽5 (Ahlenius and Hillegaart, 1986). Bar tests scores at the 15–20 min time-point were compared across groups using the non-parametric Kruskal-Wallis test, with

post hoc Dunn’s multiple comparison tests. For assessment of biomechanical measures, the values for the three time-points obtained each hour for stiffness and viscosity data were averaged. A mixed model, with a random effect for rat, was used to analyze the mean values for each hour (Stata Statistical Software: release 9; StataCorp, College Station, TX, USA). The model included a term for baseline (pre-test) values and terms for time and group. P-values for an overall difference among the groups are reported. Where these were significant P-values for differences between the groups are also reported.

RESULTS Fig. 1 shows frames taken from movies of typical vehicle, CGS21680 and raclopride bar-tests. Following vehicle injection the animal moved a paw off the bar in under 2 s, whereas following drug treatments the rats stayed in the unnatural elevated bar posture with paws motionless, for the entire 30 s shown. Analysis of group data showed a significant difference across groups (P⬍0.001, KruskalWallis test), with post hoc Dunn’s multiple comparison tests confirming that bar-test scores for both CGS21680 (3.22⫾0.97) and raclopride (1.56⫾0.88) were significantly higher than for vehicle (0⫾0; P⬍0.001, P⬍0.05, respectively), with no significant difference between CGS21680 and raclopride scores (P⬎0.05). Group data for the stiffness measure over the time course of the experiment in control, CGS21680 and raclopride tests are shown in Fig. 2A. The mixed model analysis of the three post-treatment time-points, adjusted for baseline (pre-injection values) and time, revealed a significant effect of group (P⬍0.001). Further tests confirmed significant differences between CGS21680 and vehicle (P⬍ 0.05) and between raclopride and vehicle treatments (P⬍0.001). There was no significant difference between CGS21680 and raclopride (P⫽0.27). The effect of drug treatments on viscosity is shown in Fig. 3B. The mixed model analysis of the three posttreatment time-points, adjusted for baseline (pre-injection values) and time, again revealed a significant effect of group for this measure (P⬍0.01). However, further tests showed that while the effect of raclopride was significantly (P⬍0.001) different than for vehicle, the response to CGS21680 failed to reach significance (P⫽0.091). Furthermore, comparison of CGS21680 and raclopride effects revealed a marginally significant difference (P⫽0.05).

DISCUSSION The main focus of the present study was to investigate the effect of adenosine A2A agonist treatment on measures of limb rigidity. Similar to previous reports (Heffner et al., 1989; Mandhane et al., 1997; Rimondini et al., 1997), we found that systemic administration of the selective A2A agonist CGS21680 produced catalepsy, as measured by hypokinetic performance on the bar test. Importantly, our quantification of resistance at the ankle joint in these animals also confirmed increased rigidity in the hind limb following systemic administration. This result thus confirms and extends previous work showing increased total resistance at the ankle joint following intrastriatal administration

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Fig. 3. Effect of drugs on rigidity measures. (A) Stiffness. Points show mean⫾SEM for the pre-drug measurement (Pre) and the average of three measures within the first (0 –1), second (1–2) and third (2–3) hour post-drug. CGS⫽CGS21680; Raclo⫽raclopride; * P⬍0.05, *** P⬍0.001, for mixed-model analysis post hoc comparisons between saline, and CGS21680 and raclopride groups respectively. (B) Viscosity measure; *** P⬍0.001, ns⫽not significant, for mixed-model analysis post hoc comparison between saline, and raclopride and CGS21680 groups, respectively.

of CGS21680 (Wardas et al., 1999). In that study the effect was direction-specific. The mechanism for such a directional specificity remains unclear, but may reflect differential age-related changes of connective tissue in different muscle groups around the ankle (Wolfarth et al., 1997).

Fig. 2. Derivation of stiffness and viscosity measures. (A) Raw data traces from a single experiment show measured ankle joint displacement (X), and the reactive resistance (T) during imposed sinusoidal

joint rotation, and the measured phases lag between them. Inset shows simplified mass-spring model with damping elements, showing spring and viscosity elements for only one side of the joint. Resistance (T) is a function of mass (m), spring-like elasticity (stiffness, k) and viscosity (b), of which k and b may vary as a result of drug manipulations. (B) Example angular displacement (X) vs. resistance (T) plots at different oscillation frequencies (␻) for pre-drug control, and post raclopride (Raclo). Joint stiffness was calculated as the slope of a line (dashed) through the hysteresis loop at the lowest imposed frequency (␻⫽1/3 Hz), i.e. the lowest velocity of joint movement. The viscous component for each frequency (B␻) was calculated from the phase lag, and used to calculate joint viscosity. (C) Example plot showing calculation of joint viscosity (B), from the slope of the regression through plots of B␻ as a function of ␻. Open circles pre-drug, closed circles post-raclopride.

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Thus, taken together the available data from studies in which limb tonus has been quantified suggest opposite effects of D2 and A2A receptor actions on limb rigidity, this classic sign of Parkinsonism being increased by both D2 antagonists (blocking constitutive action of endogenously released dopamine), and by A2A agonists (exaggerating the effect of endogenously released adenosine). These results are in accordance with the co-localization and known antagonistic relationship of these receptors at the cellular level (Svenningsson et al., 1999) and functionally, in expression of Parkinsonian signs (Salamone et al., 2008). In the present experiment, we separately analyzed for the first time the effect of A2A agonist on joint stiffness and viscosity. The results showed that CGS21680-induced catalepsy was accompanied by a significant increase in stiffness measured at the hind paw, that was qualitatively similar to that produced by raclopride. As noted above, increased stiffness would be considered a hallmark of Parkinsonism. On the other hand, the result for viscosity component was less clear following A2A agonist treatment. Overall, there appeared to be a trend in the same direction as for raclopride, but there was no significant difference for CGS21680 compared to vehicle, whereas there was a marginally significant difference between CGS21680 and raclopride groups. Thus, the viscous component of joint resistance appears to be less consistently affected by systemic A2A agonist than does stiffness. This is surprising, because injection of CGS21680 directly into striatum has been reported to increase short and long latency stretch reflex responses (Wardas et al., 1999), which would be expected to increase viscosity. These apparently contradictory results may reflect an opposing effect of extra-striatal receptors engaged by systemic administration in the present study. Although extrastriatal A2A receptor densities are much lower than in the striatum (Rosin et al., 1998; Svenningsson et al., 1999), a recent study using region-specific knockout of A2A receptors in mice showed that extrastriatal receptors can have functionally opposing effects to those mediated by striatal A2A receptors (Shen et al., 2008). One mechanism by which such extrastriatal A2A receptors could modulate long-loop reflexes might be through a sedative effect. Adenosine is somnogenic, and while this has been ascribed largely to the A1 adenosine receptor, infusions of CGS21680 on the surface of the basal forebrain or brainstem can also promote sleep (Basheer et al., 2004). While our animals were awake at the time of testing, a subtle sub-clinical sedative effect cannot be ruled out. An exaggerated sedative effect might also account for the findings of a previous study, which suggested that systemic administration of A2A agonist causes a relaxed, flaccid catalepsy (Rimondini et al., 1997). This was noted at a high dose (10 mg/kg, twice that used in the present study) that was sufficient to be associated with pronounced autonomic side-effects. As well as increasing activation of extra striatal receptors, higher doses would also increase occupancy of A1 receptors. Thus an important implication

of these findings is that when administered systemically, the lowest possible dose required to produce measurable catalepsy should be used, particularly in studies concerning A2A-dopamine interactions in Parkinsonism. Acknowledgments—This research was supported by the National Health Research Institute of ROC (contract no. NHRI-EX 959524E1), and the Health Research Council of New Zealand and the New Zealand Neurological Foundation. Thanks to Dr. Sheila Williams for statistical advice.

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(Accepted 29 January 2009) (Available online 4 February 2009)