Behavioral characterization of mice lacking the A3 adenosine receptor: sensitivity to hypoxic neurodegeneration - PubMed (original) (raw)

Behavioral characterization of mice lacking the A3 adenosine receptor: sensitivity to hypoxic neurodegeneration

Irina M Fedorova et al. Cell Mol Neurobiol. 2003 Jun.

Abstract

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 hotplate, 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.

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Figures

Fig. 1

Fig. 1

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.

Fig. 2

Fig. 2

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.

Fig. 3

Fig. 3

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.

Fig. 4

Fig. 4

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

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.

Fig. 6

Fig. 6

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; _a_Significantly 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

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

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