Optogenetic stimulation of the locus coeruleus enhances appetitive extinction in rats

Reviewer #1 (Public Review):

In this paper by Lui and colleagues, the authors examine the role of locus coeruleus (LC)-noradrenaline (NA) neurons in the extinction of appetitive instrumental conditioning. They report that optogenetic activation of global LC-NA neurons during the conditioned stimulus (CS) period of extinction enhances long-term extinction memory without affecting within-session extinction. In contrast, LC-NA activation during the intertrial interval doesn't affect extinction and long-term memory. They then show that optogenetic activation of LC-NA neurons doesn't induce conditioned place preference/avoidance. Finally, they assess the necessity of LC-NA neurons in appetitive extinction and find that optogenetic inactivation of LC-NA neurons during the CS period results in the enhancement of within-session extinction. The experiments are well-designed, including offset control in the optogenetic activation study. I think this study adds new insight into the LC-NA system in the context of appetitive extinction.

Strengths:
・These studies identify that the artificial activation of LC-NA neurons enhances long-term memory of appetitive extinction, while this activation can't induce long-term conditioned place aversion. Thus, optogenetic activation of LC-NA neurons can inhibit spontaneous recovery of appetitive extinction without causing long-term aversive memory.
・Optoinhibition study demonstrates the reduction of a conditioned response of within-session extinction. Therefore, LC-NA neuronal activity at the CS period of extinction could act as anti-extinction or be important for the expression of the conditioned response.

Weaknesses:
・It is unclear how LC-NA neurons behave during the CS period of appetitive extinction from this study. This weakens the importance of the optogenetic inactivation result.
・While authors manipulate global LC-NA neurons, many people find functionally heterogeneous populations in the LC. It remains unsolved if there is a specific LC-NA subpopulation responsible for appetitive extinction.

Reviewer #2 (Public Review):

This study examines the role of the Locus Coeruleus (LC)/noradrenergic (NA) system in extinction in male and female rats. The behavioural task involves three phases i) training on a discriminative procedure in which operant responding was rewarded only during the presentation of a stimulus ii) extinction iii) testing for the expression of extinction at both short (1 day) or long (7 days) delays. Targeting LC/NA cells with optogenetic in TH::Cre rats, the authors found that photoexcitation during extinction led to an increase in the expression of extinguished responding at both short and long delays. By contrast, photo inhibition was found to be without an effect.

1. In such discrimination training, Pavlovian (CS-Food) and instrumental (LeverPress-Food) contingencies are intermixed. It would therefore be very interesting if the authors provided evidence of other behavioural responses (e.g. magazine visits) during extinction training and tests.
2. In Figure 1, the authors show the behavioural data of the different groups of control animals which were later collapsed in a single control group. It would be very nice if the authors could provide the data for each step of the discrimination training.
3. Inspection of Figures 2C & 2D shows that responding in control animals is about the same at test 2 as at the end of extinction training. Therefore, could the authors provide evidence for spontaneous recovery in control animals? This is of importance given that the main conclusion of the authors is that LC stimulation during extinction training led to an increased expression of extinction memory as expressed by reduced spontaneous recovery.
4. Current evidence suggests that there are differences in LC/NA system functioning between males and females. Could the authors provide details about the allocation of male and female animals in each group?
5. The histology section in both experiments looks a bit unsatisfying. Could the authors provide more details about the number of counted cells and also their distribution along the antero-posterior extent of the LC. Could the authors also take into account the sex in such an analysis?

Reviewer #3 (Public Review):

The introduction/background is excellent. It reviews evidence showing that the extinction of conditioned responding is regulated by noradrenaline and suggests that the locus coeruleus (LC) may be a critical locus of this regulation. This naturally leads to the aim of the study: to determine whether the locus coeruleus is involved in the extinction of an appetitive conditioned response. Overall, the study is well-designed, nicely conducted and the results advance our understanding of the role of the LC in the extinction of conditioned behaviour. As such, I believe that these results will be of interest to readers. I do, however, feel that the paper would benefit from the inclusion of additional data to clarify the impact of the LC manipulations (stimulation and inhibition) on performance in the task; and some comment regarding the likely source of differences between the groups at test.