Sex Differences in Discrimination Behavior and Orbitofrontal Engagement During Context-Gated Reward Prediction

Reviewer #1 (Public Review):

Summary:
Peterson et al., present a series of experiments in which the Pavlovian performance (i.e. time spent at a food cup/port) of male and female rats is assessed in various tasks in which context/cue/outcome relationships are altered. The authors find no sex differences in context-irrelevant tasks and no such differences in tasks in which the context signals that different cues will earn different outcomes. They do find sex differences, however, when a single outcome is given and context cues must be used to ascertain which cue will be rewarded with that outcome (Ctx-dep O1 task). Specifically, they found that males acquired the task faster, but that once acquired, the performance of the task was more resilient in female rats against exposure to a stressor. Finally, they show that these sex differences are reflected in differential rates of c-fos expression in all three subregions of rat OFC, medial, lateral, and ventral, in the sense that it is higher in females than males, and only in the animals subject to the Ctx-dep O1 task in which sex differences were observed.

Strengths:
• Well-written.
• Experiments elegantly designed.
• Robust statistics.
• Behaviour is the main feature of this manuscript, rather than any flashy techniques or fashionable lab methodologies, and luckily the behaviour is done really well.
• For the most part I think the conclusions were well supported, although I do have some slightly different interpretations to the authors in places.

Weaknesses:
1. With regards to the claim (page 4 of pdf), I think I can see what the authors are getting at when they claim "Only Ctx-dep.01 engages context-gated reward predictions", because the same reward is available in each context, and the animal must use contextual information to determine which cue will be rewarded. In other words, it has a discriminative purpose. In Ctx-dep.O1/O2, however, although the context doesn't serve a discriminative purpose in the sense that one cue will always earn a unique outcome, regardless of context, the fact that these cues are differentially rewarded in the different context means that animals may well form context-gated cue-outcome associations (e.g. CtxA-(CS1-O1), CtxnoA-(CS2-O2)). Moreover, the context is informative in this group in telling the animal which cue will be rewarded, even prior to outcome delivery, such that I don't think contextual information will fade to the background of the association and attention be lost to it in the way, say Mackintosh (1975) might predict. Therefore, I don't think this statement is correct.

2. I think the results shown in Figure 1 are very interesting, and well supported by the statistics. It's so nice to see a significant interaction, as so many papers try to report these types of effects without it. However, I do wonder how specific the results are to contextual modulation. That is, should a discriminative discrete cue be used instead of each context (e.g. CS1 indicates CS2 earns O1, CS3 indicates CS4 earns O1), would female rats still be as slow to learn the discrimination?

3. Pages 8-9 of pdf, where the biological basis or the delayed acquisition of contextual control in females is considered, I find this to be written from a place of assuming that what is observed in the males is the default behaviour. That is, although the estrous cycle and its effects on synaptic plasticity/physiology may well account for the results, is there not a similar argument to be made for androgens in males? Perhaps the androgens also somehow alter synaptic plasticity/physiology, leading to their faster speed, reduced performance stability, and increased susceptibility to stress.

4. In addition, the OFC - which is the brain region found to have differential expression of c-fos in males and females in Figure 5 - is not explicitly discussed with regard to the biological mechanisms of differences, which seems odd.

Reviewer #2 (Public Review):

Summary:
A bidirectional occasion-setting design is used to examine sex differences in the contextual modulation of reward-related behaviour. It is shown that females are slower to acquire contextual control over cue-evoked reward seeking. However, once established, the contextual control over behaviour was more robust in female rats (i.e., less within-session variability and greater resistance to stress) and this was also associated with increased OFC activation.

Strengths:
The authors use sophisticated behavioural paradigms to study the hierarchical contextual modulation of behaviour. The behavioural controls are particularly impressive and do, to some extent, support the specificity of the conclusions. The analyses of the behavioural data are also elegant, thoughtful, and rigorous.

Weaknesses:
My primary concern is that the authors' claim of sex differences in context-dependent discrimination behaviour is not fully supported by their data.

First, the basic behavioural effect does not seem to replicate across experiments. The authors first show sex differences in the % time in food port and the discrimination ratio (Figures 1 and 2) such that males show better context-dependent discrimination than females (group ctx-dep O1). However, this difference is not observed in the baseline condition group in the next experiment, which investigates the effect of acute stress on context-gated reward seeking: "In Figure 4, we observe no difference between males versus females in group "ctx-dep O1".

Second, I am not fully convinced by the authors' assertion that the results are specific to the contextual modulation process. The authors' main conclusions are derived from comparing a group trained with the differential outcome procedure (group cxt-dep O1/O2) and a group with the non-differential outcome procedure (group cxt-dep O1). However, importantly, a different number of training sessions was used for ctx-dep O1/O2 and ctx-dep O1. Is it not possible that sex differences could have emerged with additional training in the cxt-dep O1/O2 group? Moreover, the authors also seem to assume that rats are not using a contextual strategy in the context-dep O1/O2 condition (i.e., rats use instead distinct context-outcome associations) but what is the evidence for this? Also, the authors argue that the impact of stress is specific to the hierarchical contextual modulation of behaviour however inspection of Figure 4A suggests that there may also be an effect of stress on the context-dependent O1/O2 group.

I also had some minor issues with how the authors interpreted some of the findings. First, it is shown that recent rewards disrupt contextual control of reward seeking in male, but not female, rats. That is, in males, prior reward increased the probability of responding on subsequent non-rewarded trials but trial history had no effect in females. How do the authors reconcile this finding with the quicker acquisition and better discrimination that is observed in males? It is not evident to me how males can have difficulty inhibiting responding to non-rewarded cues following recent reward yet still show better discrimination throughout training.

Finally, the authors argue that the contextual control over behaviour was more robust in female rats as females show less within-session variability and greater resistance to stress. What evidence is there that the restraint stress procedure causes a similar stress response in both sexes?

Reviewer #3 (Public Review):

Summary:
This manuscript reports an experiment that compared groups of rats acquisition and performance of a Pavlovian bi-conditional discrimination, in which the presence of one cue, A, signals that the presentation of one CS, X, will be followed by a reinforcer and a second CS, Y, will be nonreinforced. Periods of cue A alternated with periods of cue B, which signaled the opposite relationship, cue X is nonreinforced, and cue Y is reinforced. This is a conditional discrimination problem in which the rats learned to approach the food cup in the presence of each CS conditional on the presence of the third background cue. The comparison groups consisted of the same conditional discrimination with the exception that each CS was paired with a different reinforcer. This makes the problem easier to solve as the background is now priming a differential outcome. A third group received simple discrimination training of X reinforced and Y nonreinforced in cues A and B, and the final group was trained with X and Y reinforced on half the trials (no discrimination). The results were clear that the latter two discrimination learning procedures resulted in rapid learning in comparison to the first. Rats required about 3 times as many 4-session blocks to acquire the bi-conditional discrimination than the other two discrimination groups. Within the biconditional discrimination group, female and male rats spent the same amount of time in the food cup during the rewarded CS, but females spent more time in the food cup during CS- than males. The authors interpret this as a deficit in discrimination performance in females on this task and use a measure that exaggerates the difference in CS+ and CS_ responding (a discrimination ratio) to support their point. When tested after acute restraint stress, the male rats spent less time in the food cup during the reinforced CS in comparison to the female rats, but did not lose discrimination performance entirely. The was also some evidence of more fos-positive cells in the orbitofrontal cortex in females, but this difference was of degree.

Overall, I think the authors were successful in documenting performance on the biconditional discrimination task. Showing that it is more difficult to perform than other discriminations is valuable and consistent with the proposal that accurate performance requires encoding of conditional information (which the authors refer to as "context"). There is evidence that female rats spend more time in the food cup during CS-, but I hesitate to agree that this is an important sex difference. There is no cost to spending more time in the food cup during CS- and they spend much less time there than during CS+. Males and females also did not differ in their CS+ responses, suggesting similar levels of learning. A number of factors could contribute to more food cup time in CS-, such as smaller body size and more locomotor activity. The number of food cup entries during CS+ and CS- was not reported here. Nevertheless, I think the manuscript will make a useful contribution to the field and hopefully lead readers to follow up on these types of tasks.

One area for development would be to test the associative properties of the cues controlling the conditional discrimination, can they be shown to have the properties of Pavlovian occasion-setting stimuli? Such work would strengthen the justification/rationale for using the terms "context" and "occasion setter" to refer to these stimuli in this task in the way the authors do in this paper.

Strengths:
- Nicely designed and conducted experiment.
- Documents performance difference by sex.

Weaknesses:
- Overstatement of sex differences.
- Inconsistent, confusing, and possibly misleading use of terms to describe/imply the underlying processes contributing to performance.