Anxiety-like behaviour and adenosinergic processes in compulsive-like deer mice (Peromyscus maniculatus bairdii)

Abstract

Obsessive-compulsive disorder (OCD)1 is characterized by recurring thoughts (obsessions) and rigid repetition of certain mental or behavioral actions (compulsions) that are often associated with anxiety. In terms of neurobiology, dysfunctional connectivity in distinct cortico-limbic and cortico-striatalthalamic circuits has been shown. Two pathways, i.e. direct and indirect, relay executive signals between the prefrontal cortex and the thalamus. Whereas the direct pathway expresses dopamine D1 receptors, the indirect pathway expresses D2 receptors. Dysregulation between the activities of these pathways is proposed to underlie obsessive-compulsive symptom manifestation. Further, literature agrees that a functional imbalance between these pathways at the time of behavioral engagement, underlies repetitive behavioral output. Unfortunately, despite convincing evidence that selective serotonin reuptake inhibitors (SSRIs)2 are modestly successful in the treatment of OCD, treatment resistance remains a fundamental dilemma. Adenosine is an important neuromodulator that exerts its actions via A1 and A2 receptors. Of especial importance in this work is the A2A receptor which is located mainly in the striatum. Here, they modulate behavioral output and regulate motor control. Importantly, adenosine and dopamine receptors co-occur as A2A-D2 receptor dimers on the neuronal bodies of the indirect, behaviorally inactivating pathway. Such co-expression facilitates functional opponency, whereby adenosinergic stimulation decreases the activity of dopamine at the D2 receptor. In this regard, A2A receptor antagonists, e.g. istradefylline (ISTRA)3, may show promise as an anxiolytic and motor restorative intervention. Therefore, considering the involvement of both limbic and cortico-striatal processes in OCD as well as the proposed contribution of dysregulated direct and indirect pathway activity in the manifestation of repetitive behavior, the potential of ISTRA as an anxiolytic and anti-compulsive pharmacotherapeutic strategy was explored. When bred, housed, and reared under standard laboratory conditions, 25 - 30 % of deer mice (Peromyscus maniculatus bairdii) express large nest building (LNB)4 behavior. Being reminiscent of clinical compulsivity, LNB behavior is excessive and purposeless. Still, it remains unknown whether LNB behavior is mostly reflective of perturbations in motor control (i.e. striatal processes), or if anxiety-related cognitive processes (and thus limbic involvement) may play a role in its manifestation. This question, as well as how such associations might be modulated by anti-adenosinergic intervention, has not yet been explored and thus formed the focus of this investigation.

An initial pool of 120 deer mice of both sexes underwent initial nest building screening to select 34 NNB1- and 32 LNB2-expressing deer mice that were used in further study (ethical approval number: NWU-0042-21-A5). The selected mice were exposed to either a normal water (wCTRL)3 or vehicle control (vCTRL)4 for 28 days, lorazepam (LOR)5 for seven days (as an anxiolytic control) or ISTRA6 for 28 days. Mice were subsequently reassessed for nesting expression, this time in combination with open field anxiety testing. On the morning following the last nesting and anxiety assessment, mice were decapitated, the striatal tissue harvested on ice, and analyzed for adenosine A2A receptor expression.

The main findings of this investigation were that 1) NNB- and LNB-expressing mice showed similar behavior in an anxiogenic open field, 2) ISTRA bolstered the expression of NNB, without affecting LNB behavior or the open field behavior of either phenotype, and 3) LNB, compared to NNB behavior was associated with a noteworthy increase in the expression of lower affinity striatal A2A receptors, compared to NNB mice.

The first main finding of this investigation was that NNB- and LNB-expressing mice showed similar behaviors in an open field, indicating that NNB- and LNB-expressing mice present with similar anxietylike behavioral responses. This finding was somewhat unexpected, given our earlier results that LNBexpressing mice, assessed over the course of a full dark cycle, engage in increased risk-taking behaviors, albeit to obtain cotton wool. Nevertheless, from the present data, it can be concluded that when open field behavior is measured over the short term in a novel open field environment, NNBand LNB-expressing mice both exhibit similar levels of exploratory behavior. Thus, it can be concluded that LNB expression is not associated with generalized underlying anxiety.

The second and third main findings of this study were highly informative. Not only was it observed that ISTRA, but not control or LOR, bolstered NNB behavior, but also that LNB behavior associated with a noteworthy increase in striatal A2A receptor density (Bmax), compared to NNB behavior. While we

could not report these differences on a statistically significant level, the pharmacological and neurobiological data from this work show that naturalistic LNB1 behavior is associated with distinct indirect pathway mechanisms that are most likely founded upon lower adenosinergic, and by implication higher dopaminergic, stimulation. Therefore, it can be concluded that the act of nesting itself is intrinsically dependent on the executive balance between the direct and indirect pathways of the CSTC2 circuits. This finding paves the way for continued investigations of adenosinergic agonists in the model system as a potential ameliorative strategy.

In conclusion, our findings show that NNB3 and LNB behavior are not distinctly associated with measures of anxiety and that ISTRA4-induced changes in nesting expression are dissociated from changes in anxiety scores. Further, nesting in deer mice is directly related to striatal adenosine signaling, and LNB is founded upon a lower degree of adenosinergic A2A stimulation. Future studies should explore the effects of adenosine receptor agonists as potential ameliorative strategies.