A Biphasic and Brain-Region Selective Down-Regulation of Cyclic Adenosine Monophosphate Concentrations Supports Object Recognition in the Rat

Background: We aimed to further understand the relationship between cAMP concentration and mnesic performance. Methods and Findings: Rats were injected with milrinone (PDE3 inhibitor, 0.3 mg/kg, i.p.), rolipram (PDE4 inhibitor, 0.3 mg/ kg, i.p.) and/or the selective 5-HT4R agonist RS 67333 (1 mg/kg, i.p.) before testing in the object recognition paradigm. Cyclic AMP concentrations were measured in brain structures linked to episodic-like memory (i.e. hippocampus, prefrontal and perirhinal cortices) before or after either the sample or the testing phase. Except in the hippocampus of rolipram treated-rats, all treatment increased cAMP levels in each brain sub-region studied before the sample phase. After the sample phase, cAMP levels were significantly increased in hippocampus (1.8 fold), prefrontal (1.3 fold) and perirhinal (1.3 fold) cortices from controls rat while decreased in prefrontal cortex (,0.83 to 0.62 fold) from drug-treated rats (except for milrinone+RS 67333 treatment). After the testing phase, cAMP concentrations were still increased in both the hippocampus (2.76 fold) and the perirhinal cortex (2.1 fold) from controls animals. Minor increase were reported in hippocampus and perirhinal cortex from both rolipram (respectively, 1.44 fold and 1.70 fold) and milrinone (respectively 1.46 fold and 1.56 fold)-treated rat. Following the paradigm, cAMP levels were significantly lower in the hippocampus, prefrontal and perirhinal cortices from drug-treated rat when compared to controls animals, however, only drug-treated rats spent longer time exploring the novel object during the testing phase (inter-phase interval of 4 h)

Locomotor activity measured during the sample and the testing trails in the object recognition task.

<p>Locomotor activity measured during the sample and the testing trails in the object recognition task.</p

Effect of milrinone (0.3 mg/kg) on PP2 activities in hippocampus, prefrontal and perirhinal cortices from rats performing the object recognition task with a 4-h delay.

<p>Rats were injected with the inhibitor of PDE3 (milrinone, 0.3 mg/kg) 45 minutes before exposure then with saline or the 5-HT4 receptor agonist (RS 67333, 1 mg/kg), 30 minutes before exposure to the sample trial of the object recognition task. Immediately after the testing trial, both particulate (white bar) and soluble (black bar) fractions from the hippocampus (<b>a</b>), the prefrontal cortex (<b>b</b>) and perirhinal cortex (<b>c</b>) were isolated and were assayed for PP2 activity. PP2 activities were pmol of phosphate released by min and mg protein. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032244#s3" target="_blank">Results</a> are means ± SEM of four independent subcellular fractionations performed in triplicate. Within each subcellular compartment, # indicated significant differences of PP2 activity as compared with other treatment (#, P<0.05, ##, P<0.01, ###, P<0.001, ANOVA followed by Fisher's LSD test).</p

Time of exploration of objects measured during the sample and the testing trails in the object recognition task.

<p>Time of exploration of objects measured during the sample and the testing trails in the object recognition task.</p

Distribution of the total PP2 activities between particulate and soluble fractions from rat hippocampus, prefrontal cortex and perirhinal cortex.

<p>Values are means ± SEM.</p>§§§<p><i>P</i><0.001 (ANOVA followed by Fisher's LSD test): different from the corresponding particulate fraction.</p

Rolipram and/or RS 67333 induce a biphasic modulation of cAMP concentrations in the hippocampus, prefrontal and perirhinal cortices of rats performing an object recognition task with a 4-h delay.

<p>Rats were injected with the inhibitor of PDE4 (rolipram, 0.3 mg/kg, i.p.) and then with saline or the 5-HT4 receptor agonist (RS 67333, 1 mg/kg, i.p.), respectively 45 minutes and 30 minutes before to the sample phase of the object recognition task. Rats were euthanized before or after the sample phase, or before or after the testing phase. Cyclic AMP was extracted from the hippocampus, prefrontal and perirhinal cortices and then assayed. Cyclic AMP was expressed as pmolcAMP/mg of weight tissue. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032244#s3" target="_blank">Results</a> were means ± SEM of three independent extractions performed in duplicate. (0) indicated significant differences in comparison with other steps of the paradigm in each brain sub-region, Mann-Whitney test, P<0.05. (*) indicated a significant difference in comparison with saline treatment in each brain sub-region. (0,*, P<0.05).</p

Rolipram (0.3 mg/kg, i.p.) specifically inhibits PDE4 activities in hippocampus, prefrontal cortex and perirhinal cortex from rats.

<p>Rats were injected with the PDE4 inhibitor (rolipram, 0.3 mg/kg), and then with saline or the 5-HT4 receptor agonist (RS 67333, 1 mg/kg), respectively 45 minutes and 30 minutes before the sample phase of the object recognition task. Immediately after the testing phase, both particulate and soluble fractions from the hippocampus, the prefrontal cortex and perirhinal cortex were isolated and the particulate fraction was assayed for rolipram (10 µM)-sensitive PDE activities. Rolipram-sensitive and –insensitive PDE activities were expressed as pmolcAMPhydrolysed/min/mg protein. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032244#s3" target="_blank">Results</a> are means ± SEM of four independent subcellular fractionations performed in triplicate. Within each subcellular compartment, * indicated significant differences of PDE activity as compared with other treatment within a type of PDE activity (PDE4 or other PDE) (*, P<0.05, **, P<0.01, ANOVA followed by Fisher's LSD test).</p

Object recognition task after a 4 h-delay in saline, rolipram- or milrinone- treated rats with or without RS 67333 co-treatment.

<p>Time of exploration of the familiar and novel objects during the testing phase of the object recognition memory task of saline (n = 32), RS 67333 (n = 32), rolipram (n = 27), rolipram+RS 67333 (n = 27), milrinone (n = 27), or milrinone+RS 67333 (n = 27)-treated rats. Rats were injected with the PDE inhibitor (0.3 mg/kg, i.p.) solution 45 minutes before the sample phase and with saline or RS 67333 (1 mg/kg, i.p.) solution 30 minutes before the sample phase. Object recognition was assayed after a 4 h-delay. Values are means in s ± SEM. NS: non significant, (*) indicates a significant difference in comparison with saline treatment. (*, P<0.05, **, P<0.01, ***, P<0.001, ANOVA followed by Fisher's PLSD test).</p

Value of the index measures of discrimination in the object recognition task.

*<p>P<0.05,</p>**<p>P<0.01,</p>***<p>P<0.001, one-way ANOVA followed by Fisher LSD test, comparison with saline-treated group.</p

Expression of PDE4D proteins in the particulate and soluble fractions of rat hippocampus, prefrontal and perirhinal cortices.

<p>Particulate and soluble fractions from the rat hippocampus, the prefrontal cortex and perirhinal cortex were isolated and proteins extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032244#s2" target="_blank">Materials and Methods</a> section. The left panel shows representative immunoblots of particulate (25 µg) and soluble (25 µg) protein fractions probed with goat polyclonal human anti-PDE4D antibody in the hippocampus, prefrontal and perirhinal cortices. Arrowheads indicate the molecular weights of the immunoreactive proteins. The right panel shows quantification; the intensities of the immunoreactive bands in the particulate and soluble fractions from hippocampus, prefrontal and perirhinal cortices were determined and normalized to those of actin. The densitometry values are the mean ± SEM (n = 3).</p