Prior cocaine use disrupts identification of hidden states by single units and neural ensembles in orbitofrontal cortex

Reviewer #1 (Public review):

Summary:

In this study, the authors trained rats on a "figure 8" go/no-go odor discrimination task. Six odor cues (3 rewarded and 3 non-rewarded) were presented in a fixed temporal order and arranged into two alternating sequences that partially overlap (Sequence #1: 5⁺-0⁻-1⁻-2⁺; Sequence #2: 3⁺-0⁻-1⁻-4⁺) --forming an abstract figure-8 structure of looping odor cues.

This task is particularly well-suited for probing representations of hidden states, defined here as the animal's position within the task structure beyond superficial sensory features. Although the task can be solved without explicit sequence tracking, it affords the opportunity to generalize across functionally equivalent trials (or "positions") in different sequences, allowing the authors to examine how OFC representations collapse across latent task structure.

Rats were first trained to criterion on the task and then underwent 15 days of self-administration of either intravenous cocaine (3 h/day) or sucrose. Following self-administration, electrodes were implanted in lateral OFC, and single-unit activity was recorded while rats performed the figure-8 task.

Across a series of complementary analyses, the authors report several notable findings. In control animals, lOFC neurons exhibit representational compression across corresponding positions in the two sequences. This compression is observed not only in trial/positions involving overlapping odor (e.g., Position 3 = odor 1 in sequence 1 vs sequence 2), but also in trials/positions involving distinct, sequence-specific odors (e.g., Position 4: odor 2 vs odor 4) --indicating generalization across functionally equivalent task states. Ensemble decoding confirms that sequence identity is weakly decodable at these positions, consistent with the idea that OFC representations collapse incidental differences in sensory information into a common latent or hidden state representation. In contrast, cocaine-experienced rats show persistently stronger differentiation between sequences, including at overlapping odor positions.

Strengths:

Elegant behavioral design that affords the detection of hidden-state representations.

Sophisticated and complementary analytical approaches (single-unit activity, population decoding, and tensor component analysis).

Weaknesses:

The number of subjects is small --can't fully rule out idiosyncratic, animal-specific effects.

Comments

(1) Emergence of sequence-dependent OFC representations across learning.

A conceptual point that would benefit from further discussion concerns the emergence of sequence-dependent OFC activity at overlapping positions (e.g., position P3, odor 1). This implies knowledge of the broader task structure. Such representations are presumably absent early in learning, before rats have learned the sequence structure. While recordings were conducted only after rats were well trained, it would be informative if the authors could comment on how they envision these representations developing over learning. For example, does sequence differentiation initially emerge as animals learn the overall task structure, followed by progressive compression once animals learn that certain states are functionally equivalent? Clarifying this learning-stage interpretation would strengthen the theoretical framing of the results.

(2) Reference to the 24-odor position task

The reference to the previously published 24-odor position task is not well integrated into the current manuscript. Given that this task has already been published and is not central to the main analyses presented here, the authors may wish to a) better motivate its relevance to the current study or b) consider removing this supplemental figure entirely to maintain focus.

(3) Missing behavioral comparison

Line 117: the authors state that absolute differences between sequences differ between cocaine and sucrose groups across all three behavioral measures. However, Figure 1 includes only two corresponding comparisons (Fig. 1I-J). Please add the third measure (% correct) to Figure 1, and arrange these panels in an order consistent with Figure 1F-H (% correct, reaction time, poke latency).

(4) Description of the TCA component

Line 220: authors wrote that the first TCA component exhibits low amplitude at positions P1 and P4 and high amplitude at positions P2 and P3. However, Figure 3 appears to show the opposite pattern (higher magnitude at P1 and P4 and lower magnitude at P2 and P3). Please check and clarify this apparent discrepancy. Alternatively, a clearer explanation of how to interpret the temporal dynamics and scaling of this component in the figure would help readers correctly understand the result.

(5) Sucrose control
Sucrose self-administration is a reasonable control for instrumental experience and reward exposure, but it means that this group also acquired an additional task involving the same reinforcer. This experience may itself influence OFC representations and could contribute to the generalization observed in control animals. A brief discussion of this possibility would help contextualize the interpretation of cocaine-related effects.

(6) Acknowledge low N

The number of rats per group is relatively low. Although the effects appear consistent across animals within each group, this sample size does not fully rule out idiosyncratic, animal-specific effects. This limitation should be explicitly acknowledged in the manuscript.

(7) Figure 3E-F: The task positions here are ordered differently (P1, P4, P2, P3) than elsewhere in the paper. Please reorder them to match the rest of the paper.

Reviewer #2 (Public review):

In the current study, the authors use an odor-guided sequence learning task described as a "figure 8" task to probe neuronal differences in latent state encoding within the orbitofrontal cortex after cocaine (n = 3) vs sucrose (n = 3) self-administration. The task uses six unique odors which are divided into two sequences that run in series. For both sequences, the 2nd and 3rd odors are the same and predict reward is not available at the reward port. The 1st and 4th odors are unique, and are followed by reward. Animals are well-trained before undergoing electrode implant and catheterization, and then retrained for two weeks prior to recording. The hypothesis under test is that cocaine-experienced animals will be less able to use the latent task structure to perform the task, and instead encode information about each unique sequence that is largely irrelevant. Behaviorally, both cocaine and sucrose-experienced rats show high levels of accuracy on task, with some group differences noted. When comparing reaction times and poke latencies between sequences, more variability was observed in the cocaine-treated group, implying animals treated these sequences somewhat differently. Analyses done at the single unit and ensemble level suggests that cocaine self-administration had increased the encoding of sequence-specific information, but decreased generalization across sequences. For example, the ability to decode odor position and sequence from neuronal firing in cocaine-treated animals was greater than controls. This pattern resembles that observed within the OFC of animals that had fewer training sessions. The authors then conducted tensor component analysis (TCA) to enable a more "hypothesis agnostic" evaluation of their data.

Overall, the paper is well written and the authors do a good job of explaining quite complicated analyses so that the reader can follow their reasoning. I have the following comments.

While well-written, the introduction mainly summarises the experimental design and results, rather than providing a summary of relevant literature that informed the experimental design. More details regarding the published effects of cocaine self-administration on OFC firing, and on tests of behavioral flexibility across species, would ground the paper more thoroughly in the literature and explain the need for the current experiment.

For Fig 1F, it is hard to see the magnitude of the group difference with the graph showing 0-100%- can the y axis be adjusted to make this difference more obvious? It looks like the cocaine-treated animals were more accurate at P3- is that right?
The concluding section is quite brief. The authors suggest that the failure to generalize across sequences observed in the current study could explain why people who are addicted to cocaine do not use information learned e.g. in classrooms or treatment programs to curtail their drug use. They do not acknowledge the limitations of their study e.g. use of male rats exclusively, or discuss alternative explanations of their data.

Is it a problem that neuronal encoding of the "positions" i.e. the specific odors was at or near chance throughout in controls? Could they be using a simpler strategy based on the fact that two successive trials are rewarded, then two successive trials are not rewarded, such that the odors are irrelevant?

When looking at the RT and poke latency graphs, it seems the cocaine-experienced rats were faster to respond to rewarded odors, and also faster to poke after P3. Does this mean they were more motivated by the reward?