Locus coeruleus modulation of single-cell representation and population dynamics in the mouse prefrontal cortex during attentional switching

Reviewer #1 (Public review):

Summary:

The authors note that there is a large corpus of research establishing the importance of LC-NE projections to the medial prefrontal cortex (mPFC) of rats and mice in attentional set or 'rule' shifting behaviours. However, this is complex behavior, and the authors were attempting to gain an understanding of how locus coeruleus modulation of the mPFC contributes to set shifting.

The authors replicated the ED-shift impairment following NE denervation of mPFC by chemogenetic inhibition of the LC. They further showed that LC inhibition changed the way neurons in mPFC responded to the cues, with a greater proportion of individual neurons responsive to 'switching', but the individual neurons also had broader tuning, responding to other aspects of the task (i.e., response choice and response history). The population dynamics were also changed by LC inhibition, with reduced separation of population vectors between early-post-switch trials, when responding was at chance, and later trials when responding was correct. This was what they set out to demonstrate, and so one can conclude they achieved their aims.

The authors concluded that LC inhibition disrupted mPFC "encoding capacity for switching" and suggest that this "underlie the behavioral deficits."

Strengths:

The principal strength is the combination of inactivation of LC with calcium imaging in the mPFC. This enabled detailed consideration of the change in behavior (i.e., defining epochs of learning, with an 'early phase' when responding is at chance being compared to a 'later phase' when the behavioral switch has occurred) and how these are reflected in neuronal activity in the mPFC, with and without LC-NE input.

Weaknesses:

Methodologically, some improvement could be made in terms of the statistical descriptions. Supplementary Figure 2: For the peripheral CNO, the 'control group' (saline) was n=4 and the test group (CNO), n=5. For the central CNO, the test group was n = 8 and the control was n = 7. The authors explain that the group sizes were not statistically determined and mice were assigned to groups 'arbitrarily', but why did they not at least make the group sizes equal?

In Figure 1 (e), given the small sample size, it would be helpful if all the data points were included on the bar charts. As a t-test was performed on only the ED stage of the test, seeing all the data points would reassure that there would not have been a statistically significant 'improvement' in the ID stage in the group given mPFC CNO. It would also be helpful to give effect sizes for all statistical tests.

Reviewer #2 (Public review):

Summary:

The authors were building on prior research linking cortical norepinephrine in a test of attentional set shifting. They extended prior research by assessing the effects of excitatory or inhibitory DREADDs prior to the test of attentional set shifting.

Strengths:

The use of DREADDs in the previously validated test of attentional set shifting improves temporal control of corticopetal, noradrenergic pathways during behavior. While mice typically require multiple intradimensional shifts to form an attentional set, the subjects in the current study perform a variant of the task similar to that used in humans, improving the translational validity of the work.

Weaknesses:

A critical piece of evidence needed to support the behavioral claim that mice form an attentional set is a statistically significant difference between the number of trials to reach criterion at the intradimensional vs. the extradimensional stage of the task. Based on prior literature, this could be done as a planned comparison, which would improve the power to detect differences beyond that found using an HSD test. An additional methodological ambiguity is the amount of time between the administration of CNO and the performance of the ED. As reported, it seems likely that the DREADDS were impacting performance at multiple stages of the test.

Overall, the authors seem to have achieved their aims, but have failed to provide critical statistical support for claims made.

The work is likely to be of interest to the burgeoning number of scientists investigating the role of cortical norepinephrine in cognitive flexibility.

Reviewer #3 (Public review):

Summary:

Nigro et al examine how the locus coeruleus (LC) influences the medial prefrontal cortex (mPFC) during attentional shifts required for behavioral flexibility. Specifically, they propose that LC-mPFC inputs enable mice to shift attention effectively from texture to odor cues to optimize behavior. The LC and its noradrenergic projections to the mPFC have previously been implicated in this behavior. The authors further establish this by using chemogenetics to inhibit LC terminals in mPFC and show a selective deficit in extradimensional set-shifting behavior. However, the study's primary innovation is the simultaneous inhibition of LC while recording multineuron patterns of activity in mPFC. Analysis at the single neuron and population levels revealed broadened tuning properties, less distinct population dynamics, and disrupted predictive encoding when LC is inhibited. These findings add to our understanding of how neuromodulatory inputs shape attentional encoding in mPFC. However, several issues somewhat limit the overall impact and interpretation of the results.

Strengths:

The more naturalistic set-shifting task used in the study is a major strength, and its implementation in freely-moving animals is very useful. The inclusion of localized suppression of LC-mPFC terminals is also a strength that builds confidence in the specificity of their behavioral effect. Moreover, the combination of chemogenetic inhibition of LC while simultaneously recording neural activity in mPFC with miniscopes is state-of-the-art. The authors apply analyses to population dynamics, in particular, that can advance our understanding of how the LC modifies patterns of mPFC neural activity. The authors show that neural encoding at both the single-cell level and the population level is disrupted when LC is inhibited. They also show that activity is less able to predict key aspects of the behavior when the influence of LC is disrupted. This is quite interesting and adds to a growing understanding of how neuromodulatory systems sharpen the tuning of mPFC activity.

Weaknesses:

There are some concerns about tying the results to noradrenergic circuit activity. The authors use a DBH-Cre mouse line, but the histology images provided are low resolution, and surprisingly, there appears to be little overlap between HM4Di expression and TH immunostaining. It is unclear what explains this, but without further confirmation, it is hard to be sure whether the manipulation selectively impacts a specific LC population. While the authors are generally conservative in relating their findings to norepinephrine (NE) signaling, it is still implied that this is likely. But even if HM4Di is expressed specifically in DBH+ LC neurons, there is no confirmation that NE release is suppressed, and these neurons may release other neurotransmitters, including glutamate and dopamine. In the absence of careful controls, it is important to recognize that effects may or may not be due to LC-mPFC NE.

Another weakness is that the behavior of miniscope mice is not shown. These experiments make up the bulk of the study, including the most significant results (Figures 2-4). Interpreting the chemogenetics + imaging results without this data is more challenging and relies on the assumption that they were affected similarly to an animal from Figure 1. More fundamentally, the imaging analyses are entirely from the extradimensional shift session. Showing similar analyses from the intradimensional shift (IDS) session would confirm that test group mice do not exhibit broadened tuning prior to injecting CNO and would help to establish whether the observed changes are to some feature of activity that is specific to extradimensional shifts. The ideal experiment would also include a separate group of animals with LC suppression during the IDS, which would show whether the observed effects are specific to an extradimensional shift and might explain behavioral effects.

There are also some weaknesses in how the single neuron encoding data is analyzed and presented. First, the corresponding methods section is insufficient to fully understand how selectively tuned neurons were classified. The authors perform ROC analysis for the period 0 - 5s before choice to reveal choice-tuned neurons. It would be useful to know what proportion of the total neurons this represents, and whether this includes neurons with activity that is significantly increased, decreased, or both. Further, insufficient detail is provided to be able to understand how neurons are further classified into 'choice', 'history', and 'switch' categories, or what percentage of ROC-identified neurons fall into each category (only % of total neurons is provided).

Finally, there are some concerns about lumping all the identified neurons together (as in Figure 2F). The miniscope experiments include very few mice (n=4 controls, n=5 test), and effects may be driven by only 1 or 2 subjects. Also, plotting the data on a per-animal basis would help to better understand the effects in greater detail. Overall, the results are interesting, but these weaknesses limit the strength and specificity of the claims that can be made.