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3-Iodothyronamine (T1AM) is an endogenous primary amine probably produced through thyroid hormone metabolism (Scanlan, 2009; Piehl et al., 2011). T1AM circulates in healthy rodents and humans (Saba et al., 2010; Galli et al., 2012) but it concentrates in tissues, particularly in the liver and brain (Saba et al., 2010). Recently, several studies have explored the consequences of administering T1AM directly into the brain. T1AM injected i.c.v. rapidly induces metabolic effects, including a reduction of body temperature (Doyle et al., 2007), modification of food intake and hyperglycaemia (Manni et al., 2012). T1AM effects were not linearly related to the dosage and depended on the animal species and administration route (Dhillo et al., 2008; Klieverik et al., 2009). We have recently reported that 1.32 μg·kg−1 T1AM injected i.c.v. into fasted mice produced hypophagia and hyperglycaemia, while it reduced plasma fT3 and fasting-induced c-fos activation (Manni et al., 2012). Both hypophagia and fT3 reduction were not linearly related to T1AM doses and they were prevented by in vivo treatment with clorgyline, an inhibitor of oxidative deamination, the major pathway of T1AM catabolism (Saba et al., 2010), suggesting the involvement of rapid desensitizing targets and/or interference with neuromediators producing opposing effects on behaviour. In fact, electrophysiological experiments suggest that T1AM affects the response to catecholamines and other neurotransmitters, acting as a specific inhibitor of dopamine and noradrenaline re-uptake and of monoamine transport into synaptic vesicles (Snead et al., 2007). Therefore, T1AM might be regarded as a neuromodulator.
In the present paper, we investigated whether T1AM is able to produce specific neurological effects such as the modification of memory acquisition and pain threshold in mice, and whether these effects are modified under conditions of MAO inhibition by clorgyline and whether they were associated with changes in triiodothyroxine (T3) and thyroxine (T4) brain levels.
With this aim, we evaluated the behaviour of mice injected i.c.v. with T1AM (0.13, 0.4, 1.32 and 4 μg·kg−1) in the passive avoidance test and in the novel object recognition task, as well as the effects of T1AM on pain threshold, exploratory activity and plasma glycaemia. In parallel experiments, the activation of typical signalling proteins involved in memory acquisition and pain perception, including pERK, pAkT, c-fos and pCREB, were measured in specific brain areas. Brain levels of T3 and T4 and of T1AM were also determined following injection of T1AM at the lowest effective dose.
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We report for the first time that T1AM given i.c.v. behaves as a memory enhancer in mice. This effect was achieved by stimulating curiosity without modifying locomotor activity or producing any analgesic effect. At the dosages active on memory, T1AM also turned out to be hyperalgesic. Interestingly, T1AM was similarly potent at stimulating memory and curiosity, suggesting that increased exploratory activity is part of the memory-enhancing effect, whereas it appeared to be more potent at producing hyperalgesic and hyperglycaemic effects. The latter therefore represents a sort of ‘fingerprint’ of T1AM pharmacological properties.
In cell cultures and in isolated hearts, T1AM showed a short half-life largely due to cellular uptake and oxidative deamination to 3-iodothyroacetic acid (Saba et al., 2010). This observation was confirmed in our experimental setting. By 30 min after its i.c.v. injection, the brain T1AM concentration had decreased from a nominal value of 231 pmol·g−1 to 13 pmol·g−1, consistent with its degradation and/or transport into the systemic circulation (Manni et al., 2012). Therefore, we wondered whether T1AM metabolite(s) might contribute to its effects. To ascertain the role of oxidative deamination, we repeated the passive avoidance and the hot plate tests in animals pretreated with clorgyline, which has been reported to increase T1AM concentration and to abolish the production of deaminated derivatives, particularly of 3-iodothyroacetic acid (Saba et al., 2010; Manni et al., 2012). Under these conditions, in the passive avoidance test, the response to T1AM was markedly reduced and did not reach the threshold of statistical significance. In the pain threshold experiments, a significant response was still observed, but the dose–response relationship was modified, as the dose of 0.13 μg·kg−1, which was ineffective in the absence of clorgyline, turned out to be hyperalgesic, while this effect disappeared at higher doses. These results suggest either that T1AM acts on rapid desensitizing targets, or that some of its effects are mediated by deaminated derivatives, such as 3-iodothyroacetic acid. Although the latter could not be detected in brain tissue, it should be pointed out that the assay procedure was optimized to detect T1AM, and had a low sensitivity for acid derivatives such as 3-iodothyroacetic acid (Saba et al., 2010).
The interpretation of our findings is further complicated by the observation that clorgyline per se induced a significant reduction in the pain threshold. This might imply that endogenous T1AM and/or its derivatives play a physiological role in pain sensitivity but might also be explained by the hypothesis that the response to T1AM is antagonized by different aminergic systems. Notably, the effect of T1AM on feeding was also modulated by clorgyline (Manni et al., 2012), while behavioural effects of MAO inhibition have been reported in different experimental models (Whitaker-Azmitia et al., 1994). These issues deserve further investigation, and in particular, it would be interesting to evaluate the effects of i.c.v. injections of 3-iodothyroacetic acid.
T1AM has been reported to interact with trace amine-associated receptor 1 (TA1 receptor), which is expressed together with other members of the TA receptor family in different brain regions (Zucchi et al., 2006). Interestingly, TAAR genes are located in a region that is associated with psychiatric disorders in linkage studies (Revel et al., 2011). However, the role of TA receptors could not be directly demonstrated in the present study because specific TA receptor antagonists are not available. Different receptors might also be involved, including α2 adrenoceptors, which have been implicated in the pancreatic response to T1AM (Regard et al., 2007).
ERK1/2 is a member of the family of MAPKs. The corresponding signalling cascade has been reported to activate cAMP-responsive element binding protein (CREB) and other transcription factors, inducing the synthesis of proteins that are required for the stabilization of new memories (Kida et al., 2002; Pittenger et al., 2002) and the regulation of long-term synaptic plasticity (Roberson and Sweatt, 1999). It is well known that pERK2 and pAkt are cross-talking signals involved in memory (Chen et al., 2008) and that pERK2 and pAkt exert opposite effects on the expression and activity of several transcription factors essential for the synthesis of new proteins necessary for memory stabilization, including CREB and c-fos (Peng et al., 2011). However, in this context, we were unable to detect modifications of pERK, pCREB or c-fos levels in any of the brain regions analysed following injection of 1.32 μg·kg−1 T1AM, while we detected significantly higher pERK levels after exposure to lower T1AM doses, namely 0.04 and 0.13 μg·kg−1. This finding suggests that ERK1/2 signalling might be quickly and selectively activated by low T1AM doses, whereas rapid desensitization occurs at higher doses. Memory acquisition requires activation of receptor cascades and synthesis of new proteins involved in memory retention, and ERK phosphorylation appears to be a very early event, which precedes gross behavioural effects, but its specific causal role remains to be determined. The observation that T1AM activates pERK in brain regions such as the amygdala and hippocampus might also be related to the hyperalgesic response (Schicho et al., 2005; Ji et al., 2009; Liu et al., 2012), which was elicited by low T1AM doses. The signalling cascade leading to the hyperalgesic effect is unknown, but T1AM might inhibit the release of analgesic mediators (Hu et al., 2007) on the basis of its putative effect on α2 adrenoceptors (Regard et al., 2007). In any case, the hyperalgic and pro-learning effects cannot be accounted for by changes in thyroid hormone concentration (Guasti et al., 2007), which were not detected in brain tissue after T1AM injection.
It has already been reported that i.c.v. injection of T1AM modulates insulin and/or glucagon secretion and produces a rise in plasma glycaemia (Manni et al., 2012). In the present work, we observed that hyperglycaemia occurs even at the dosage of 0.13 μg·kg−1, which is 10 times lower than that previously used (Manni et al., 2012). There is no evidence that moderate hyperglycaemia may produce behavioural effects similar to those reported in the present investigation. However, this issue deserves further investigation. In particular, it would be interesting to assess the behavioural effects of T1AM on the diabetic/hypothyroid mouse.
To the best of our knowledge, this is the first report indicating that the central effects of T1AM include the regulation of complex behavioural functions involved in learning and pain perception. These actions were associated with an increase in local T1AM concentration of about one order of magnitude, suggesting a novel potential physiological role of endogenous T1AM and/or its deaminated derivative(s). Thyroid hormones are essential for the development of mammalian brain and maintenance of optimal cognitive ability in different periods of life (Bauer et al., 2008). In adulthood, thyroid dysfunction leads to neurological and behavioural abnormalities, including memory impairment. Adult-onset hypothyroidism is also associated with clinically relevant cognitive dysfunctions such as psychotic behaviour, hallucinations, confusion and learning defects (Rivas and Naranjo, 2007). Central hypothyroidism has been reported in patients with Alzheimer's disease (Sampaolo et al., 2005) and the analysis of different experimental models suggests that the effects on cognition rely on hippocampal modifications. In the present work, we demonstrated that T1AM, an endogenous compound related to thyroid hormones, stimulates the acquisition of memory in the mouse and that this effect does not involve significant modifications of brain thyroid hormone levels. It should be noted, thyroid hormone levels found in the brains of our mice were similar to those demonstrated by Escobar-Morreale et al. (1996) and Pinna et al. (2002). Due to these novel effects, our results suggest that pharmacological administration of T1AM might be useful in neurodegenerative and endocrine disorders associated with memory deficits.