DREADD-mediated modulation of locus coeruleus inputs to mPFC improves strategy set-shifting

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Abstract

Appropriate modification of behavior in response to our dynamic environment is essential for adaptation and survival. This adaptability allows organisms to maximize the utility of behavior-related energy expenditure. Modern theories of locus coeruleus (LC) function implicate a pivotal role for the noradrenergic nucleus in mediating switches between focused behavior during periods of high utility (exploit) versus disengagement of behavior and exploration of other, more rewarding opportunities. Two modes of activity in LC neurons have been characterized as elements in an Adaptive Gain Theory (AGT) of LC function. In this theory, during periods of accurate and focused behavior, LC neurons exhibit task-related phasic bursts. However, as behavioral utility wanes, phasic activity is suppressed and baseline (tonic) impulse activity increases to facilitate exploration. Our experiments sought to exogenously induce an elevated pattern of activity in LC neurons and their medial prefrontal cortical (mPFC) targets to test the tenets of the AGT. This theory posits that tonic activation immediately following a rule change should increase exploration and thereby improve performance on a set-shifting task. Indeed, DREADD mediated stimulation of LC terminals within mPFC decreased trials to reach criterion. However, this effect resulted from improved application of the new rule once the original rule is jettisoned rather than earlier disengagement from the old, ineffective strategy. Such improvements were not seen with global manipulation of LC, consistent with the view that LC-mediated exploration involves specific sub-circuits targeting mPFC. These findings extend our understanding of the role of LC in PFC and flexible behavior.

Introduction

Appropriate modification of ongoing behavior is critical to an organism’s ability to adapt and thrive in a dynamic environment. When the utility of a behavior is high, maintenance of that behavior is adaptive to exploit a rewarding opportunity. However, it becomes advantageous to disengage from a behavior when utility wanes to explore potentially more rewarding opportunities. The Adaptive Gain Theory proposes that activity of noradrenergic neurons in locus coeruleus (LC) plays a central role in mediating these trade-offs by adaptively regulating gain throughout target networks (Aston-Jones and Cohen, 2005a, Aston-Jones and Cohen, 2005b), a viewpoint consistent with other suggestions (Bouret and Sara, 2005, Corbetta et al., 2008, Yu and Dayan, 2005).

Recordings of LC neurons in monkeys have shown that accurate performance on a visual discrimination task is related to low baseline (tonic) levels of activity with task-related phasic bursts closely following the detection of a target stimulus (Aston-Jones et al., 1994, Clayton et al., 2004, Rajkowski et al., 1994, Usher et al., 1999). However, when a subject is not attending to the task and commits errors frequently, task-related phasic bursts are attenuated while tonic activity becomes elevated (Aston-Jones et al., 2000, Aston-Jones et al., 1999, Usher et al., 1999). We have recently shown that elevations in tonic LC activity increase decision noise and facilitate disengagement from cognitive tasks (Kane et al., 2017). In rats, phasic activity has been repeatedly shown to facilitate detection of and orienting towards environmental stimuli (Aston-Jones and Bloom, 1981, Bouret and Sara, 2004, Devilbiss and Berridge, 2006, Waterhouse et al., 1998). Hence, noradrenergic LC neurons are thought to play an important role in adaptively orienting animals in response to changes in reward opportunities in their environment.

Noradrenergic activity also plays a key role in facilitating cognitive flexibility in situations requiring shifts in attention between different aspects of complex stimuli. To assess attentional or strategy set-shifting, tasks are conducted wherein animals are initially required to learn a particular discrimination rule (e.g. stimulus association, sequence responding, spatial discrimination) to obtain a reward. At a predetermined point the rule is shifted, requiring the animal to modify its behavior to continue to receive reward; this is referred to as a strategy set-shift and used as a translationally-relevant measure of cognitive flexibility (Birrell and Brown, 2000, Haluk and Floresco, 2009, Newman et al., 2008, Tait et al., 2007). The medial prefrontal cortex (mPFC) plays a key role in facilitating extradimensional shifts (EDS), a form of strategy set-shift), as do LC inputs to mPFC (Birrell and Brown, 2000, Joel et al., 1997, Ragozzino et al., 1999). Specifically, lesions of the rat LC dorsal noradrenergic bundle (DNAB), the source of noradrenaline to mPFC, or selective lesions of noradrenergic terminals in mPFC increase the number of trials required to reach criterion performance on the EDS (Tait et al., 2007, McGaughy et al., 2008). Deficits induced by NE deafferentation of mPFC can be rescued by systemic administration of the noradrenaline reuptake blocker atomoxetine (Newman et al., 2008). These studies therefore indicate a critical role for noradrenergic mPFC/LC systems in cognitive flexibility.

Although lesions or pharmacological interference with noradrenergic signaling degrades cognitive flexibility, it remains unclear whether activation of LC neurons can facilitate shifting from one strategy to another. In this regard, the small size and compact location of LC rendered cell-specific stimulation of these NE cells in behaving animals technically difficult until reports utilizing new techniques for cell-specific stimulation and inhibition (Carter et al., 2010, Hickey et al., 2014, Vazey and Aston-Jones, 2014, Vazey and Moorman, 2018). The advent of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) (Armbruster et al., 2007, Farrell and Roth, 2013) enables selective expression of synthetic receptors within LC-NE neurons that can be stimulated by administration of clozapine n-oxide (CNO). In the present study we used the synthetic dopamine-beta hydroxylase promoter, PRSx8, to drive expression of the hM3Dq DREADD selectively in NE neurons of LC (Vazey & Aston-Jones, 2014). In a previous report, we found that a local microinfusion of CNO into LC expressing hH3Dq elevated baseline activity and decreased inter-spike interval in vivo without altering phasic bursting activity (Vazey & Aston-Jones, 2014). In the present study, we activated these receptors locally within either LC-NE terminals in mPFC (after axonal transport), or in the LC nucleus, via microinjection of CNO in rats performing a modified version of an operant-based strategy set-shifting task (Floresco, Block, & Tse, 2008). mCherry control (non-DREADD) subjects were used in each experiment to control for potential off-target effects of CNO or clozapine metabolites (Gomez et al., 2017). This approach enabled us to examine effects of activating either the entire LC noradrenergic projection system, versus stimulating only the LC-NE input to PFC, on cognitive flexibility involving shifts between different discrimination rules.

Section snippets

Animal care and surgery

All methods used were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Medical University of South Carolina Institutional Animal Care and Use Committee. Adult male Long-Evans rats (250–300 g) were obtained from Charles River Laboratories (Raleigh, NC). Animals were housed in a temperature and humidity-controlled room under a reverse 12 h light/dark cycle (lights on at 18:00 h) with water available ad libitum. All

Selective expression of viral gene products in LC-NE neurons

Selective expression of hM3Dq (Fig. 2A) and control mCherry reporter protein (Fig. 2B) in NE neurons of LC was achieved with PRSX8-regulated viral vectors. Of the animals expressing hM3Dq in LC, cannulae were accurately placed within or just lateral to LC in 22 animals (Fig. 2C, closed circles) or within PL/IL cortex in 9 animals (Fig. 2D, closed circles). Of animals expressing the control reporter mCherry, cannulae were accurately placed within or just lateral to LC in 9 animals (Fig. 2C, open

Discussion

Here, we showed that stimulation of LC-NE inputs to mPFC increases behavioral flexibility during strategy set-shift. These results complement previous reports using lesion methods (McGaughy et al., 2008, Tait et al., 2007), and provide direct evidence that selective stimulation of LC-NE terminals in mPFC facilitates EDS performance (Lapiz and Morilak, 2006, Newman et al., 2008). In contrast, similar stimulation of noradrenergic cell bodies in LC did not facilitate performance, but instead

Conclusion

Overall, this study reveals that stimulation of LC terminals in mPFC is sufficient to improve performance on a strategy set-shifting task. This effect is regionally specific as it is only witnessed when CNO administration is confined within PFC and not when administered within the LC. Future study into site-specific mechanisms of hM3Dq receptors as well as the regionally specific contributions of other LC targets to this behavior would be helpful in determining the mechanisms that underlie this

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