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. Author manuscript; available in PMC: 2011 Jul 28.
Published in final edited form as: Cell Mol Neurobiol. 2003 Jun;23(3):431–447. doi: 10.1023/a:1023601007518

Behavioral Characterization of Mice Lacking the A3 Adenosine Receptor: Sensitivity to Hypoxic Neurodegeneration

Irina M Fedorova 1, Marlene A Jacobson 2, Anthony Basile 1,3,4, Kenneth A Jacobson 1
PMCID: PMC3145360  NIHMSID: NIHMS31419  PMID: 12825837

SUMMARY

1. The potential neuroprotective actions of the A3 adenosine receptor (A3AR) were investigated using mice with functional deletions of the A3AR (A3AR−/−) in behavioral assessments of analgesia, locomotion, tests predictive of depression and anxiety, and the effects of mild hypoxia on cognition and neuronal survival.

2. Untreated A3AR−/− mice were tested in standard behavioral paradigms, including activity in the open field, performance in the hot-plate, tail-flick, tail-suspension, and swim tests, and in the elevated plus maze. In addition, mice were exposed repeatedly to a hypoxic environment containing carbon monoxide (CO). The cognitive effects of this treatment were assessed using the contextual fear conditioning test. After testing, the density of pyramidal neurons in the CA1, 2, and 3 subfields of the hippocampus was determined using standard histological and morphometric techniques.

3. A3AR−/− mice showed increased locomotion in the open field test, elevated plus maze (number of arm entries) and light/dark box (number of transitions). However, they spent more time immobile in two different tests of antidepressant activity (Swim and tail suspension tests). A3AR−/− mice also showed evidence of decreased nociception in the hot-plate, but not tail-flick tests. Further, A3AR−/− mice were more vulnerable to hippocampal pyramidal neuron damage following episodes of carbon monoxide (CO)-induced hypoxia. One week after exposure to CO a moderate loss of pyramidal neurons was observed in all hippocampal subfields of both wild-type (A3AR+/+) and A3AR−/− mice. However, the extent of neuronal death in the CA2–3 subfields was less pronounced in A3AR+/+ than A3AR−/− mice. This neuronal loss was accompanied by a decline in cognitive function as determined using contextual fear conditioning. These histological and cognitive changes were reproduced in wild-type mice by repeatedly administering the A3AR-selective antagonist MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate 1 mg/kg i.p.).

4. These results indicate that pharmacologic or genetic suppression of A3AR function enhances some aspects of motor function and suppresses pain processing at supraspinal levels, while acting as a depressant in tests predictive of antidepressant action. Consistent with previous reports of the neuroprotective actions of A3AR agonists, A3AR−/− mice show an increase in neurodegeneration in response to repeated episodes of hypoxia.

Keywords: Adenosine receptor, neurodegeneration, knockout mice, analgesia, depression

INTRODUCTION

The four subtypes of adenosine receptors (ARs), A1, A2A, A2B, and A3, are widely distributed throughout the body (Fredholm et al., 2001). Activation of the G protein-coupled receptors A1 and A3 inhibits adenylate cyclase activity, while activation of the A2A and A2B subtypes stimulates this enzyme. Stimulation of A1, A2B, and A3 subtypes also activate phospholipases (Feoktistov et al., 2002; Parsons et al., 2000). In the periphery, A3AR activation by either endogenous adenosine or exogenously administered agonists appears to be cardioprotective (Parsons et al., 2000), an effect mediated in cultured myocytes by phospholipase D. However, this cell-protective function of A3AR may not be universally observed. The cardioprotective, hemodynamic, and toxic effects of A3AR agonists appears to be species-dependent (Lasley et al., 1999). In rodents, acute activation of A3ARs increases the release of proinflammatory agents such as histamine and TNFα (Ramkumar et al., 1993; Sajjadi et al., 1996; Salvatore et al., 2000; van Schaick et al., 1996). Moreover, deletion of the A3AR in mice enhanced adenosine-stimulated coronary blood flow (Talukder et al., 2002) and generally improved cardiac tolerance to ischemia (Guo et al., 2001; Harrison et al., 2002). These results suggest that, in some species, inhibition of A3AR activity may protect tissues, possibly by suppressing proinflammatory processes.

The functions of the A3AR in the central nervous system (CNS) appear to be equally complex. The precise density and localization of A3AR in the brain remains unclear, due in part to the absence of selective, high affinity ligands or nucleotide probes (Rivkees et al., 2000). Nonetheless, there is some evidence for the expression of the A3AR in the hippocampus, thalamus, and hypothalamus (Yaar et al., 2002). Moreover, electrophysiological and biochemical evidence supports the localization of A3AR in the rat hippocampus (Dunwiddie et al., 1997; Macek et al., 1998) and cortex (Brand et al., 2001), where they variously exert a direct neuroinhibitory action, or cause excitation by suppressing presynaptically located mGlu III autoreceptors (Macek et al., 1998). Functional evidence also supports the presence of A3AR in the brain. The A3AR agonist N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) depresses behavior (Jacobson et al., 1993), and chronic treatment was found to be neuroprotective in the gerbil model of ischemia (von Lubitz et al., 1994).

While the lack of high affinity A3AR specific radioligands and other pharmacological tools have impeded characterization of the localization and activity of the A3AR in general, the function of the A3AR in the cardiovascular system was substantially illuminated by the development of the A3AR null (A3AR−/−) mouse. Similarly, A3AR−/− mice have the potential for providing the functional equivalent of A3 antagonist administration by mitigating the question of A3AR ligand specificity. Therefore, we performed behavioral and provocative functional characterizations of A3AR−/− mice in order to elucidate the function of the A3 receptor in the CNS.

MATERIALS AND METHODS

MRS 1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenyl-pyridine-5-carboxylate) was purchased from Sigma-RBI (St. Louis, MO). This adenosine receptor antagonist was dissolved in 100% dimethylsulfoxide(DMSO) as a 5-mM stock solution. Injection vehicles were made by diluting the stock into Alkamuls-620, then phosphate buffered saline, pH 7.4, to yield a final composition of 5%/20%/75%. All other chemicals were from standard commercial sources and of analytical grade.

Animals

Two genotypes of mice were used: adenosine A3 receptor knockout (A3AR−/−) mice developed by Merck Research Laboratories, and wild-type (A3AR+/+) control mice. A description of the generation of an earlier strain of A3AR−/− mice by Merck Research Laboratories has been described (Salvatore et al., 2000). These A3AR−/− mice were backcrossed against C57Bl/6 mice (Taconic Farms, Germantown, New York) for 12 generations to obtain a congenic line. Male mice (6–8 weeks of age, approximately 30 g) were used in all studies. The mice were housed in polypropylene cages with free access to food and water in an AALAC-certified vivarium. The vivarium was maintained on a 12-h light: 12-h dark cycle (lights on at 7:00) with a room temperature of 22 ± 1°C and relative humidity level of 50 ± 5%. All studies were approved by the NIH Institutional Animal Care and Use Committee and were conducted in compliance with the Guide for the Care and Use of Laboratory Animals.

Behavioral Studies

All behavioral studies were performed between 9 a.m. and 5 p.m. in mice acclimated to the testing room.

Tests of Nociception

The sensitivity of mice to painful stimuli was assessed using the tail-flick and hot-plate tests. In the tail-flick test, the tail of an unrestrained mouse was placed under the focused beam of a halogen projection lamp. The intensity of the lamp was adjusted so that the average latency before tail movement from the beam path by A3AR+/+ mice was approximately 5 s, with a cutoff latency of 10 s. The hot plate test involved the placement of mice onto a 55°C plate (Columbus Instruments, Columbus, Ohio). The latency to the first incidence of hind-paw shaking or licking was recorded.

Spontaneous Locomotor Activity

Mice were placed into the center of an open field apparatus (40 × 40 cm; Columbus Instruments, Columbus, OH) under dim lighting. Motor activity parameters (distance traveled, number of vertical and stereotypic movements, ambulating and resting times) were monitored and recorded over a 30-min period. Monitoring was begun immediately after the mice were placed in the open field activity monitor.

Anxiety Tests

The apparatus used for the light–dark transition test consisted of a cage (25 × 40 × 20 cm) divided in two by a black partition with a small central opening connecting the chambers. One chamber had white walls and was brightly illuminated, whereas the other chamber was black and dimly illuminated. Mice were placed into the lit compartment and allowed to move freely between the two chambers for 5 min. The number of transitions between the two compartments, the time spent in each chamber, and the latency to the first transition were recorded. The elevated plus-maze consisted of two open (45 × 5 cm) and two enclosed arms of the same size, with 15 cm high walls. The arms were constructed of black acrylic radiating from a central platform (5 × 5 cm) to form a plus sign. The entire apparatus was elevated 30 cm above the floor. The test was initiated by placing each mouse on the central platform facing one of the open arms. The number of entries into the open and closed arms and the time spent on the open and closed arms were recorded over a 5-min test period.

The Forced-Swim Test

Each mouse was placed individually into a Plexiglas cylinder (20 cm height × 10 cm diameter) filled with water (22°C) to a height of 10 cm (Porsolt et al., 1979). The total time the mouse spent immobile was recorded over a 6-min test period. Mice were judged immobile when they ceased swimming and remained floating motionless in the water, making only those movements necessary to keep their heads above the surface of the water.

The Tail-Suspension Test

This test is based on the observation that mice suspended by their tails show alternating periods of agitation and immobility (Steru et al., 1985), and is predictive of antidepressant activity. Mice were suspended using adhesive tape placed 20 mm from the tip of the tail and were kept 20 cm from the nearest object. An observer blinded to the mouse genotype determined the cumulative duration of immobility over the 6-min duration of the test.

Contextual Fear Conditioning

Contextual, hippocampus-dependent memory was assessed in mice using the conditioned fear paradigm (Sterneck et al., 1998). During the initial, context-conditioning phase, the animal learns to associate an aversive stimulus with the novel environment or the context in which it is placed. In this task, a novel environment (the distinct new context the animal is placed in) is paired with an electric shock administered on the training day. On the test day (24 h later), if the mouse has learned the context–shock association, then the same environmental context will elicit fear, resulting in a total lack of movement or freezing. The amount of time a mouse spends immobile or frozen on a given testing day is indicative of the degree of fear conditioning. Each mouse was placed in a test chamber (San Diego Instruments) and allowed to explore freely for 2 min. The conditioned stimulus (80 dB tone) was presented for 30 s followed by a mild (2 s, 0.5 mA) foot shock. Subsequently, the mouse was allowed to explore for 2 min. A second pairing of tone and shock was presented after this 2-min period, followed by another 1 min of exploration. The mouse was then removed from the chamber and returned to its home cage. The mouse was returned to the test chamber after 24 h for a 3-min period and the presence of freezing behavior recorded (context test). Contextual fear conditioning experiments were conducted on the seventh and eighth day after the second day of CO exposure.

Carbon Monoxide-Induced Neurodegeneration

Carbon monoxide exposure was carried out as previously described (Nabeshima et al., 1991). Each mouse was placed in an acrylic cylinder (10 cm × 3 cm radius) with gas entry and exit barbs. The subject was exposed to pure CO gas until it began gasping (about 20 s after exposure at the rate of 45 mL/min), then was rapidly removed to room air. Animals were exposed for 2 days, five times a day, with 1 h between each exposure. Core body temperature was maintained at 38°C by placing the mice on a heating blanket immediately after the first CO exposure and for up to 2 h after the last exposure to avoid CO hypothermia, with subsequent blunting of hypoxic neurodegeneration (Ishimaru et al., 1991).

MRS 1523 (1 mg/kg, i.p.) was administered for 5 days, with the last 2 days of administration overlapping the first 2 days of CO exposure. Control mice received vehicle.

Histology

Following the behavioral studies, mice were euthanized by AALAC-approved methods, their brains removed and placed overnight in 20% sucrose containing 0.025% DMSO. The next morning, the brain regions were blocked, and the samples flash-frozen in isopentane at −20°C. Serial 20 μm thick sections of the entire hippocampus were cut, and mounted, stained with 1% cresyl violet, destained, differentiated, and coverslipped. Morphometric analysis of CA1-3 pyramidal neurons was performed by manually counting neurons in every fifth section of cresyl violet-stained hippocampus at 400× magnification (Kustova et al., 1999). Area measures of the pyramidal neuron layer (CA1-3) of the entire hippocampus were made using digitally acquired images of hippocampal sections at 4× magnification, stacked and summed to yield the final volume measurements (Meta-Morph, Universal Imaging, Downingtown, PA).

Statistical Analysis

Data are expressed as mean ± standard error for the indicated n. Statistical analyses were performed using either a t test or one-way ANOVA followed by Bonferroni’s Multiple Comparison Test (Prism, GraphPAD Software, San Diego, CA).

RESULTS

Gross physical examination of A3AR−/− mice revealed an 8% greater body weight than age-matched A3AR+/+ mice (22.0 ± 0.4 g, n = 12 vs. 23.8 ± 0.4 g, n = 11; A3AR+/+ and A3AR−/− respectively, P < 0.01, t test), and a 1.6% lower core body temperature (37.3 ± 0.1°C, n = 12 vs. 36.7 ± 0.1°C, n = 11; A3AR+/+ and A3AR−/− respectively, P < 0.01, t test). Moreover, A3AR−/− mice demonstrated a decrease in the threshold of pain sensitivity as assessed in the hot-plate test, with a 40% increase in the latency of the hind-paw response (P < 0.05, t test, Fig. 1(A)). However, the latency to movement in the tail-flick test was unchanged (2.3 ± 0.1 s vs. 2.2 ± 0.1 s).

Fig. 1.

Fig. 1

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A3AR−/−mice show alterations in nociception and locomotion. (A) A3AR−/− mice showed a significant increase in the latency to lick a hind-paw in the hot-plate test (P < 0.05 vs. A3AR+/+ mice, t test, n = 12. (B) Spontaneous locomotor activity in the open field was significantly increased in A3AR−/− mice (Genotype effect, P < 0.01, F = 11.7, 1 df, two-way ANOVA, n = 10). (C) No difference between the genotypes was noted in the number of vertical movements made in the open field. Each bar represents the mean ± SEM.

Locomotor activity in a novel environment was measured in the open field. A3AR−/− mice showed a small but significant increase in the distance traveled in the open field (Fig. 1(B)). Interestingly, there was no significant difference between the genotypes in the number of vertical movements (Fig. 1(C)). Moreover, A3AR−/− and A3AR+/+ mice performed equally well in the rotarod test (115 ± 20 s vs. 103 ± 21 s, n = 11 both groups; A3AR+/+ and A3AR−/− respectively).

Basal levels of anxiety were assessed in the elevated plus-maze test. Although there was a trend toward increased time spent in the open arms by A3AR−/− mice, this did not reach significance (Fig. 2(A)). However, the number of open- and closed-arm entries made by A3AR−/− mice were significantly higher (113, 67%, respectively) than A3AR+/+ mice (Fig. 2(B) and (C)). A3AR−/− and A3AR+/+ mice did not differ in the time spent in the lit compartment in the light–dark transition test (120 ± 10 s for A3AR+/+, n = 12 and 140 ± 9 s for the A3AR−/− mice, n = 12). Nonetheless, A3AR−/− mice showed a significant, 95% increase in the number of transitions between the two compartments (18.7 ± 2.0, n = 12 vs. 33.2 ± 2.9, n = 12; A3AR+/+ and A3AR−/− mice, respectively, P < 0.01, t test).

Fig. 2.

Fig. 2

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A3AR−/− mice show increased locomotor activity in the elevated plus-maze test. Although A3AR−/− mice did not spend significantly more time in the open arms than A3AR+/+ mice (A), they made more entries into open (B) and closed arms (C). **Significantly different from A3AR+/+ mice, P < 0.01, t test. Each bar represents the mean ± SEM of n = 11 mice.

The behavior of the A3AR−/− and age-matched A3AR+/+ mice in the Porsolt forced swim test and tail suspension test was also investigated. In the tail suspension test, the A3AR−/− mice spent significantly more time immobile than A3AR+/+ mice, consistent with an increased level of “behavioral despair” (Fig. 3(A)). Increased immobility (49%) was also observed in A3AR−/− mice in the forced swim test (Fig. 3(B)).

Fig. 3.

Fig. 3

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A3AR−/− mice show evidence of an increase in behavioral despair. The duration of immobility by A3AR−/− mice was increased in both the tail suspension test (A) and forced swim tests (B). *P < 0.05, t test. Each bar represents the mean ± SEM of n = 11 mice.

Finally, the sensitivity of A3AR−/− mice to hypoxic neurodegeneration was investigated using an in vivo model of CO-induced delayed amnesia and neuronal damage (Maurice et al., 1994, 1999; Nabeshima et al., 1991). The influence of CO exposure on the locomotion of A3AR−/− mice was observed in the open field test. CO exposure decreased the locomotion of both genotypes of mice (Fig. 4(A)), but the relative decrease was approximately 85% greater in A3AR−/− than A3AR+/+ mice (Fig. 4(B)). CO-induced changes in cognition were also examined using a contextual fear-conditioning paradigm. Basal, pre-CO exposure rates of contextual learning were similar in mice of both genotypes (the ratio of the number of photobeam interruptions during the context test relative to the conditioning test was 19.6 ± 2.3% in A3AR+/+ mice, n = 14; and 19.1 ± 1 5% in A3AR−/− mice, n = 12). In contrast, A3AR−/− mice did not perform as well as A3AR+/+ mice after CO exposure (Fig. 5). During the conditioning phase, A3AR−/− mice showed almost the same number of photobeam interruptions as A3AR+/+ mice, consistent with equal freezing times (Fig. 5(A), conditioning). Strikingly, A3AR−/− mice interrupted the photobeams more frequently during context testing conducted 24 h after conditioning (Fig. 5(A), test-context), with their freezing time in response to shock-associated context being lower than A3AR+/+ mice. This difference is clearer when performance is presented as the ratio of photobeam interruptions during context testing to the number of photobeam interruptions during the conditioning period × 100 (Fig. 5(B)). When the retention test was conducted 1 month after conditioning, A3AR−/− mice showed a deficit in memory for the environmental context associated with an aversive stimulus. This was manifested as a 67% increase in the number of photobeam interruptions (200 ± 21 for A3AR+/+, n = 8; 340 ± 18 for A3AR−/−, n = 9, P < 0.001, t test). Administration of the A3AR antagonist MRS 1523 to A3AR+/+ mice for 5 days (protocol 2) resulted in similar changes in contextual fear conditioning as the deletion of the A3AR (Fig. 5), with the number of photobeam interruptions decreased during conditioning, but enhanced in context testing.

Fig. 4.

Fig. 4

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Effect of carbon monoxide exposure on the locomotor activity of A3AR−/− and A3AR+/+ mice. (A) CO exposure reduced the distance traveled in the open field by both A3AR−/− and A3AR+/+ mice. *Significantly different from A3AR+/+ mice, P<0.05. **Performance significantly different from respective pre-CO groups, P < 0.01. All two-way ANOVA, Bonferroni’s post-hoc analysis. (B) The relative CO-induced decrease in distance traveled in the open field was greater in A3AR−/− than A3AR+/+ mice. **Significantly different from A3AR+/+ mice, P < 0.01, t test. (n = 9 in both groups).

Fig. 5.

Fig. 5

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The influence of CO exposure on contextual fear conditioning. (A) The number of photobeam interruptions counted during the context testing of CO-treated mice 24 h after conditioning (Test-context Phase) was significantly higher in A3AR−/− mice compared to wild-type animals The same effect was observed in wild-type mice treated with MRS 1523 relative to vehicle treated mice. There was no significant difference between the groups during the conditioning phase. (B) The relative changes in freezing amongst the different groups is better observed as the ratio of test-context to conditioning performance. The percentage increase in photobeam interruptions was significantly increased in A3AR−/− mice and wild-type mice treated with MRS 1523. * P < 0.05, ANOVA, Bonferroni’s test, n = 8–11, ** P < 0.01, ANOVA, Bonferroni’s test, n = 11–14.

Finally, we examined whether the post-hypoxic cognitive deficits displayed by the A3AR−/− mice were associated with neuronal death in the CA1-3 subfields of the hippocampus. Histological studies showed that 7 days after exposure to CO, a moderate loss of pyramidal neurons was observed in all subfields of the hippocampus. Closer examination of the CA2 and CA3 regions of the hippocampus revealed that neuronal density in the CO-exposed mice was significantly more pronounced in the A3AR−/− mice than A3AR+/+ (Figs. 6 and 7) (P < 0.05, one-way ANOVA). Consistent with the enhanced loss of pyramidal neurons in A3AR−/− mice, A3AR+/+, mice administered the A3AR antagonist MRS 1523 showed a decrease in neuronal density in CA2–3 pyramidal neuron subfields.

Fig. 6.

Fig. 6

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Effect of CO treatment on hippocampal pyramidal neurons in the CA2–3 subfields. Treatment groups included näive wild type and A3AR−/− mice, and CO-exposed animals (wild type, A3AR−/− and wild type mice after vehicle administration and after A3R antagonist MRS 1523 administration). There was no difference in neuron density between näive wild type and A3AR−/− mice. However, after CO treatment, all groups showed a lower neuron density than untreated mice, neuronal death after CO exposure being more pronounced in A3AR−/− mice than in wild type mice. *Significantly different from respective näive groups, P < 0.05; aSignificantly different from CO-treated A3AR+/+mice, P < 0.05; **Significantly different from A3AR+/+mice, ANOVA and Tukey’s test. Each bar represents the mean ± SEM of data from 6 to 12 mice.

Fig. 7.

Fig. 7

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Delayed death of CA2–3 pyramidal cells in the hippocampus 7 days after CO exposure. Representative photomicrographs of 20 μm coronal sections of cresyl violet-stained hippocampal CA2–3 subfields are shown.

DISCUSSION

The modulatory role of A1 and A2AARs in the CNS is well established, and behavioral consequences of the deletion of these receptor subtypes have been described (Gimenez-Llort et al., 2002). In contrast, the A3AR have been implicated in a variety of peripheral organ system functions, including regulating cellular components of the immune system (Ramkumar et al., 1993; Sajjadi et al., 1996; Salvatore et al., 2000; Shepherd et al., 1996), cardiovascular function (Cerniway et al., 2001; Guo et al., 2001; Lasley et al., 1999; van Schaick et al., 1996; Zhao et al., 2000), and apoptosis (Appel et al., 2001; Di Iorio et al., 2002). However, understanding of their function(s) in the CNS has been impeded, both by a lack of specific ligands and the low density of receptors (Rivkees et al., 2000). In an attempt to understand the role of the A3AR in CNS function, we have utilized mice with a deletion of the A3AR receptor (A3AR−/− mice) in a battery of behavioral tests. Moreover, given evidence of a cardioprotective action of the A3AR (Parsons et al., 2000), we investigated the potential influence of these receptors on neuronal survival following a hypoxic insult.

Our initial characterization of the behavioral effects of the functional deletion of the A3AR in mice showed that A3AR−/− mice manifested a decreased sensitivity to some painful stimuli, as evidenced by the increase in latency in the hot plate, but not tail-flick test. This observation reflects a decrease in the supraspinal processing and “recognition” of painful stimuli, and may be consistent with preliminary observations of the localization of A3AR in thalamic nuclei, but not in the spinal cord (Yaar et al., 2002), where they may play a role in processing nociceptive information. Interestingly, significant increases in some aspects of motor function were also observed in A3AR−/− mice. Evidence for a slight increase in locomotor activity was observed in three different tests: activity in the open field; number of arm entries in the elevated plus maze; and number of transitions in the light/dark box. This change in motor activity appears to be selective, as there was no increase in the number of vertical movements, or evidence of “ataxia,” as defined using the rotarod, and argues against a nonselective increase in motor activity resulting from the process of generating transgenic mice. While the brain region(s) involved in this hyperlocomotion remains unknown, the disinhibition of cortical neurons resulting from the deletion of the A3AR (Brand et al., 2001) may play a role, and is consistent with the substantial depression of motor activity observed after administration of the A3AR agonist IB-MECA (Jacobson et al., 1993).

In addition to the evidence for a potential role of the A3AR in motor function and nociception, A3AR may regulate other behavioral activities. Initial interpretation of the performance of the A3AR−/− mice in the elevated plus maze and light/dark box suggests a reduction in their level of anxiety or fearfulness, as evidenced by the increase in exploratory activity. Closer examination suggests that these results may reflect the increased motor activity of these mice, as there was no significant increase in the amount of time spent in the open arms of the plus maze, or in the illuminated side of the light/dark box. While AR antagonists have been proposed as antidepressants (El Yacoubi et al., 2001), A3AR−/− mice showed an increase in the amount of time spent immobile in two tests of behavioral depression. This response is not adequately explained by a decrease in motor activity, particularly in view of the increased locomotion expressed by this genotype. A3AR have been found to modulate hippocampal serotonin levels, possibly by regulating release and/or reuptake (Okada et al., 1999). Deletion of these receptors may reduce the levels of free serotonin or catecholamines, thereby acting as a behavioral depressant and increasing immobility time in the forced-swim and tail-hang tests, which are predictive of antidepressant actions (Porsolt et al., 1979; Steru et al., 1985).

In contrast with the above studies, a role for the A3AR in aspects of neuroprotection was supported by studies with the A3AR−/− mice. While the A1and A2AARs are associated with the protective functions of adenosine, decreasing energy demand while increasing energy supply (Jacobson, 1998), respectively, the role of the A3AR has been more difficult to generalize, as both protective and damaging effects have been noted (Appel et al., 2001; Cerniway et al., 2001; Fishman et al., 2002). Repetitive exposure of mice to CO gas induces a long-lasting but delayed amnesia measured 1 week after exposure (Nabeshima et al., 2001), accompanied by dose-dependent death of hippocampal pyramidal neurons (Ishimaru et al., 1991). This relatively mild insult was used to quantify changes in neuronal vulnerability to hypoxia that may occur in the absence of the A3AR. Therefore, we characterized the behavior of a strain of A3AR−/− mice in a wide range of behavioral tests with or without mild ischemic damage induced by CO exposure. In the case of repeated brief exposure to CO, we found that mice lacking the A3AR are more vulnerable than control animals to hippocampal damage following hypoxia. By histological examination 1 week after the exposure to CO, a moderate neuronal loss in all regions of the hippocampus was observed in all mice, but the degree of neuronal death in the CA2–3 subfields was more pronounced in A3AR−/− mice than in wild type. The hypoxic damage also impacted cognition, with CO-induced hypoxia causing a decline in cognitive function in a test of contextual fear conditioning that was more pronounced in A3AR−/− mice. While this increase in test-context motor activity may reflect the increase in general locomotor activity expressed by A3AR−/− mice in select environments such as the open field, plus-maze and light–dark box, there was apparently no difference between the genotypes in performance in the contextual fear conditioning test before CO exposure. Moreover, CO exposure appeared to reduce the motor activity of the A3AR−/− mice to the level of the wild-type mice. An additional factor which may explain the apparent decrease in cognitive function manifested by CO exposed A3AR−/− mice is the increased threshold for nociception indicated by the hot-plate test. This would effectively reduce the intensity of the aversive stimulus which the environmental context is associated with, potentially slowing the rate and efficiency of learning in these mice. Nonetheless, even the evidence for changes in nociception in the A3AR−/− mice is unclear, as it reflects performance in the hot plate, but not tail-flick test.

While pre-CO exposure control testing in the contextual fear conditioning assay was performed to help mitigate the influence of potentially confounding factors, additional cognitive testing using paradigms that either do not require a nociceptive input, or extensive locomotor activity (e.g., operant conditioning) may be required before any conclusions regarding the A3AR, learning and memory in both intact and lesioned mice can be reached. This task may be eased by the observation that the apparent neuronal vulnerability observed in the A3AR−/− mice was duplicated by blockade of the receptor. Repeated administration of the A3AR-selective antagonist MRS 1523, which is selective for A3AR in both primates and rodents, resulted in a greater degree of neuronal damage and impairment of contextual memory than after vehicle administration. The role of the A3AR in neuroprotection and cognition may be more extensively tested in the future by chronic administration of MRS 1523 in concert with various hypoxia/ischemia lesioning paradigms, and/or with specific tests of cognition. Thus, in acute global brain ischemia it appears that either pharmacological blockade or genetic deletion of the A3AR in the mouse is detrimental. Although the damage incurred by the CO treatment was milder than in other models of global ischemia, such as bilateral occlusion of the carotid arteries in gerbils (von Lubitz et al., 1994), differences based on the presence or absence of the A3AR were evident. This implies that the levels of adenosine in the brain during hypoxia/ischemia are sufficient to activate the A3AR.

In summary, the A3AR−/− mouse reveals a number of CNS functions where the A3AR plays a role, including nociception, locomotion, behavioral depression, and neuroprotection. The finding that deletion of the A3AR has a detrimental effect in a model of mild hypoxia suggests the possible use of A3AR agonists in the treatment of ischemic, degenerative conditions of the CNS. A3AR agonists are already under development for other clinical indications, such as antineoplastics (Fishman et al., 2002). As the development of these pharmacotherapeutic agents progresses (Jacobson et al., 1997), the utility of pharmacologically regulating CNS activities via the A3AR may become more apparent.

ACKNOWLEDGMENTS

We thank Dr John Daly and Josh Blaustein (NIDDK) for their many helpful discussions.

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