Sex-specific behavioral and thalamo-accumbal circuit adaptations after oxycodone abstinence

Reviewer #1 (Public review):

Summary:

This manuscript by Alonso-Caraballo et al, is a novel piece of work that examines the impact of oxycodone self-administration on neural plasticity within paraventricular thalamic (PVT) to nucleus accumbens shell (Shell) pathway - two regions shown to play a key role in cue-induced drug seeking on their own, and whether this plasticity varies based on abstinence period and biological sex.

Strengths:

The authors show using a clinically relevant long-access model of opioid self-administration promotes dependence and acute withdrawal in both male and female rats. During subsequent cue-induced relapse tests at 1 or 14-days following the conclusion of self-administration, data show that while both male and females demonstrate drug-seeking behavior at both time points, females show a further elevation in responding on day 14 versus day 1 that is not observed in the males. When accounting for past work showing elevations in drug seeking in males after 30 days, these data indicate that craving-induced relapse for opioids may develop faster and may be more pronounced in females compared to males.

These behavioral findings were paralleled by use of ex vivo acute slice electrophysiology and circuit-specific ex vivo optogenetics to examine the impact of oxycodone self-administration on synaptic strength within the paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) pathway(s). Data support a time-dependent but sex independent strengthening of glutamatergic signaling at PVT-to-NAcSh medium spiny neurons (MSNs) that is only present following a relapse test at 14 days post abstinence in males versus females, providing the first evidence that opioid self-administration and/or cue-induced drug-seeking augments this pathway. Using an extensive set of physiological measures, the authors show that this increased synaptic strength reflects a upregulation of presynaptic release probability. Further, this upregulation of excitatory signaling aligned temporally with an increase in MSN excitability, as assessed by increases in action potential firing frequency. Finally, the authors provide the first evidence that similar to other inputs to the NAcSh, PVT projections innervate both MSN as well as local interneurons, promoting a GABA-A specific feedforward inhibitory circuit. Interestingly, unlike direct excitatory inputs to MSNs, no changes were observed ostensibly within this feedforward circuit, highlighting a selective enhancement of excitatory drive and output of MSNs with protracted abstinence.

Overall, these data highlight a potential role for heightened synaptic strength within the PVT-NAcSh pathway in cue-induced relapse behavior during protracted abstinence and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals.

Weaknesses:

Overall, the experimental approach and data provided appear rigorous and support their overall conclusions and achieve their goal of understanding how opioid self-administration impacts synaptic strength within the PVT-NAcSh pathway. Although not undermining these data, there are a few potential weaknesses that reduce the impact of the work. For example, the inability to directly assess whether cue-induced drug-seeking is in fact augmented compared to daily intake during self-administration in the maintenance face only permits the authors to denote that reexposure to cues and the context is sufficient to promote active lever pressing without demonstrating whether seeking behavior is in fact elevated further during a cue test. This is notably understandable as drug available sessions were 6-hours versus a 1hour relapse test. Importantly, it is clearly demonstrated that drug seeking is higher on average in female mice after 14 days versus 1 day.

With regard to interpretation of electrophysiology findings, the lack of inclusion of an abstinence only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test. While much literature has shown that drug induced adaptations in the NAc requires a post drug period for plasticity to measurably emerge, studies have also shown that re-exposure to heroin-associated cues following abstinence seemingly "reverses" increases in cell excitability in prelimbic-NAc pyramidal neurons (Kokane et al., 2023) and that depotentiation of morphine-induced increases in synaptic strength in the NAc shell can be depotentiated by drug re-exopsure -- an effect also observed with cocaine re-exposure (Madayag et al., 2019). Notably, the lack of effect at 14 but not 1 day supports the likelihood that the relapse test does not in fact influence the plasticity within the PVT-NAcSh circuit.

While the lack of effect on AMPAR:NMDAR ratio and rectification indices do support the notion that enhanced EPSC amplitudes in input-output curves do not reflect a change in AMPAR subunit expression (i.e., increased GluA2-lacking receptors that exhibit inward rectification at depolarized potential) nor a change in postsynaptic sensitivity to glutamate, without direct assessment of AMPAR-specific and NMDAR-specific input-output curves, it doesn't definitively exclude the possibility that both AMPA and NMDA receptor currents are being upregulated, thus negating an observable change in postsynaptic strength.

Overall, these findings provide novel insight into how the PVT-NAcSh pathway is altered by opioid self-administration and whether this is unique based on abstinence period and sex. Importantly, these were the primary objectives stated by the author. Data highlight a potential role for the observed adaptations in relapse behavior and identify a potential therapeutic target during abstinence to reduce relapse risk in abstaining individuals. However, it should be noted that no causal link is demonstrated without experiments to reduce/prevent relapse.

Comments on revisions:

The authors addressed previous concerns brought up, specifically by clarifying data interpretation as well as text modifications related to potential caveats of these interpretations. However, I recommend that the title be changed to not focus on sex differences to avoid misunderstanding. The authors should also address the lack of difference physiologically compared to the behavior as a caveat more clearly in the discussion (i.e. likely suggests this isn't the pathway driving the difference).

Reviewer #2 (Public review):

Summary:

This is an interesting paper from Alonso-Caraballo and colleagues that examines the influence of opioid use, acute and prolonged abstinence, and sex on cue-induced relapse and paraventricular thalamus (PVT) to nucleus accumbens shell (NAcSh) medium spiny neurons circuit physiology. The study presents a valuable finding that following prolonged, but not acute abstinence from oxycodone self-administration, female rodents exhibit higher relapse rates to drug paired cues. Additionally, the study presents the useful finding that prolonged abstinence increased PVT-NAcSh MSN synaptic strength in both sexes, an effect that is likely due to presynaptic adaptations. While the evidence to support these two findings is solid, further experiments are required to determine the functional role of the PVT-NAcSh MSN circuit in relapse following prolonged oxycodone abstinence, and the mechanism underlying the heightened relapse vulnerability in females in this model of opioid use disorder.

Strengths:

The paper is interesting, well written and presented, and the experiments are well designed and conducted. The revised analysis of spike count data that models the hierarchical structure of the data is appropriate to overcome low animal numbers and the potential for oversampling. The authors are transparent in reporting the results related to this analysis in figure 5 and acknowledge the study is underpowered to confirm the trend of increased intrinsic excitability in male MSNs following prolonged oxycodone analysis.

Weaknesses:

A major weakness of this study is the disconnect between the behavioral and neurophysiological data reported. While a striking sex difference in relapse-like behavior is observed, there are no statistically significant sex differences in any of the neurophysiological data reported. Moreover, without an experiment to functionally test the role of the PVT-NAc projection in relapse-like behavior following prolonged oxycodone these two arms of the study seem divorced.

While the authors don't directly conclude that the PVT-NAc MSN circuit is required for relapse following prolonged oxycodone abstinences, in the introduction the authors state they aim to test the hypothesis that increased synaptic strength in PVT-NAcSh projections are necessary for drug-seeking. This study does not include the required experiments to test this hypothesis.

Impact:

The topic is of interest to the field of substance use disorders and gives solid evidence for the need to consider targeted therapeutics aimed at relapse prevention in opioid use disorder.

Reviewer #3 (Public review):

Summary:

Alonso-Caraballo et al. use behavioral testing and ex vivo patch-clamp electrophysiology combined with circuit-specific optogenetic stimulation of PVT terminals to examine how oxycodone self-administration and abstinence duration shape cue-induced relapse and PVT-NAcSh synaptic transmission in male and female rats. In the revision, the authors reanalyzed intrinsic excitability using nested hierarchical GLMMs, acknowledged the low power in the male prolonged-abstinence group, and expanded the discussion of relevant PVT-NAc literature. These changes improve the manuscript. That said, most of the revisions are textual and the main experimental gap remains. Both sexes show increased oxycodone seeking compared to saline at 14 days, but only females show a time-dependent incubation from 1 to 14 days, and the PVT-NAcSh synaptic strengthening is the same in both sexes. Nothing in the revision brings those two observations closer together. The excitability data also come from NAcSh MSNs with no confirmation of PVT connectivity, which limits what circuit-specific conclusions can be drawn. The study is a solid characterization of abstinence-related synaptic changes in this pathway, but some of the conclusions still go further than the data allow.

Strengths:

The behavioral characterization is thorough and well-executed, covering self-administration, somatic withdrawal, and cue-induced relapse across two abstinence durations in both sexes. The sex-specific escalation in oxycodone seeking from 1 to 14 days in females but not males is a clear and compelling finding. The use of circuit-specific ex vivo optogenetics to isolate PVT terminal inputs onto NAcSh neurons is a genuine methodological strength, and the demonstration of feedforward inhibitory recruitment through local GABAergic interneurons adds meaningful novelty to the circuit characterization. The reanalysis of intrinsic excitability using nested hierarchical GLMMs appropriately accounts for the non-independence of cells recorded within the same animal and is a real improvement over the original approach. The expanded discussion of prior PVT-NAc work, particularly the more accurate treatment of Keyes et al. (2020) and Paniccia et al. (2024), better situates the findings within the existing literature.

Weaknesses:

The core limitation of the study remains unchanged after revision. The PVT-NAcSh synaptic strengthening after prolonged abstinence is statistically indistinguishable between sexes, while females but not males show a time-dependent escalation in oxycodone seeking from 1 to 14 days of abstinence. The Discussion proposes hormonal modulation or differences in upstream inputs as possible explanations, but none of these are tested and the gap is left unresolved. The intrinsic excitability recordings come from NAcSh MSNs with no confirmation that those neurons receive direct PVT input, which was raised in the original review, acknowledged in the revision, and not experimentally addressed. The male prolonged-abstinence excitability trend has approximately 20% statistical power and is non-significant, yet the Discussion interprets it as a potential neuroadaptation that could facilitate signal flow through the PVT-NAcSh circuit and contribute to relapse, which goes well beyond what the data support. The failure to distinguish between D1 and D2 MSNs remains a significant limitation given that cell-type-specific plasticity at PVT-NAc synapses has been shown to be directly relevant to opioid seeking in prior work. Finally, the Conclusion builds a mechanistic framework around D2 MSNs, PV interneurons, and D1 MSNs that is drawn from studies using different drugs or experimental designs, and none of these cell-type-specific mechanisms are tested in the present experiments.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

(1) Although not undermining these data, there are a few potential weaknesses that reduce the impact of the work. For example, the inability to directly assess whether cue-induced drug-seeking is in fact augmented compared to daily intake during self-administration in the maintenance face only permits the authors to denote that re-exposure to cues and the context is sufficient to promote active lever pressing without demonstrating whether seeking behavior is in fact elevated further during a cue test. This is notably understandable as drug available sessions were 6-hours versus a 1-hour relapse test. Importantly, it is clearly demonstrated that drug seeking is higher on average in female mice after 14 days versus 1 day.

We agree that the current design does not allow us to directly assess whether cue induced drug-seeking is augmented relative to the average self-administration intake. However, this comparison was not a question examined in the manuscript and was not an intended interpretation of the data. Our analyses and interpretations focused on comparisons between saline and oxycodone groups tested under identical cue-induced relapse conditions. While it does not change or contradict the reviewer’s point, we would also like to clarify that the relapse test was 2 hours long.

(2) With regard to the interpretation of electrophysiology findings, the lack of inclusion of an abstinence-only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test.

The inclusion of an abstinence-only control group would have been required to definitively dissociate synaptic changes driven by abstinence alone from those arising from an interaction between abstinence and re-exposure to the operant context during the relapse test. In the present study, electrophysiological recordings were intentionally performed 30 to 45 minutes following the relapse test to capture synaptic modifications associated with cue-induced drug-seeking after abstinence. Accordingly, we interpret these findings as reflecting the neural state following relapse rather than abstinence alone, and we have revised the text accordingly to clarify this point.

(3) With regard to the interpretation of electrophysiology findings, the lack of inclusion of an abstinence-only group does not permit interpretations to parse out whether observed increases in synaptic strength (or the lack of) reflect abstinence or an interaction between abstinence period and re-exposure to the operant chamber, as slices were taken 30-45 min post relapse test. While much literature has shown that drug-induced adaptations in the NAc require a post-drug period for plasticity to measurably emerge, studies have also shown that re-exposure to heroin-associated cues following abstinence seemingly "reverses" increases in cell excitability in prelimbic-NAc pyramidal neurons (Kokane et al., 2023) and that depotentiation of morphine-induced increases in synaptic strength in the NAc shell can be depotentiated by drug re-exposure - an effect also observed with cocaine re-exposure (Madayag et al., 2019). Notably, the lack of effect at 14 but not 1 day supports the likelihood that the relapse test does not in fact influence the plasticity within the PVT-NAcSh circuit.

We thank the reviewer for highlighting relevant literature showing that drug or cue re exposure can modify or reverse drug-induced plasticity in NAc-related circuits. We want to clarify that, in our dataset, synaptic changes in the PVT-NAcSh pathway are seen after 14 days of abstinence, but not after 1 day. Therefore, the lack of effect at the earlier time point and its appearance after extended abstinence support the idea of time-dependent plasticity. Although electrophysiological recordings were taken soon after the relapse test, this temporal pattern argues against relapse testing alone as the primary driver of the observed synaptic changes. We have updated the text to clarify this point.

(4) While the lack of effect on AMPAR:NMDAR ratio and rectification indices do support the notion that enhanced EPSC amplitudes in input-output curves do not reflect a change in AMPAR subunit expression (i.e., increased GluA2-lacking receptors that exhibit inward rectification at depolarized potential) nor a change in postsynaptic sensitivity to glutamate, without direct assessment of AMPAR-specific and NMDAR-specific input output curves, it doesn't definitively exclude the possibility that both AMPA and NMDA receptor currents are being upregulated, thus negating an observable change in postsynaptic strength.

We agree that unchanged AMPAR/NMDAR ratios and rectification index suggest against altered AMPAR subunit composition or simple postsynaptic sensitivity changes. Although receptor-specific input-output analyses would be necessary to definitively rule out proportional increases in both AMPA and NMDA receptor currents, we have updated the manuscript to clarify that our conclusions are limited to the synaptic measures we obtained. The revised text now states that acute or prolonged abstinence “might have no detectable postsynaptic effects as assessed by these synaptic measures” at PVT-NAcSh synapses.

Reviewer #2 (Public review):

(5) While this paper is certainly interesting, and well-written, and the experiments seem to be well performed, the behavioral and physiological effects observed are somewhat divorced. Specifically, what accounts for the heightened relapse in females? Since no opioid-related sex differences were observed in PVT-NAcSh neurophysiology, it is unclear how the behavioral and neurophysiological data fit together. Furthermore, the lack of functional manipulation of PVT-NAcSh circuitry leaves one to wonder if this circuit is even important for the behavior that the authors are measuring. I would be more positive about this study if the authors were able to resolve either of the two issues noted above.

A key challenge in circuit-based studies of motivated behavior is connecting circuit-level plasticity to complex, sex-dependent behavioral phenotypes. In this study, we do not mean to imply that synaptic plasticity within the PVT-NAcSh projection alone explains the increased relapse seen in females. Instead, our electrophysiological data indicate that this projection experiences time-dependent, abstinence-dependent changes in synaptic strength, offering important insights into when and where circuit-level adaptations may occur. We also believe that the lack of obvious sex differences in PVT-NAcSh synaptic strength does not rule out this circuit's role in sex-specific behavior. Growing evidence suggests that sex differences in relapse and motivated behaviors may stem from different modulation of shared circuits (for example, via ovarian hormones, neuromodulatory tone, or upstream inputs), rather than from significant differences in baseline synaptic properties within a given projection. Regarding circuit relevance, extensive previous research has identified the PVTNAcSh pathway as a critical regulator of cue-induced reward seeking and relapse. Our findings expand on this by showing that this projection displays abstinence-dependent synaptic strengthening after oxycodone self-administration. Although functional manipulation of this circuit is needed to confirm its causal role, such experiments were beyond the scope of this study.

(6) There are insufficient animals in some cases. For example, in Figure 4, the Male Saline 14-day abstinence group (n = 3 rats) has less than half of the excitability as compared to the Male Saline 1-day abstinence group (n = 7 rats). This is likely due to variance between animals and, possibly, oversampling. Thus, more rats need to be added to the 14-day abstinence group. Additionally, the range of n neurons/rat should be reported for each experiment to ensure readers that oversampling from single animals is not occurring.

We appreciate the reviewer's concern regarding the number of animals and the potential for oversampling. We take this concern seriously and have substantially revised our statistical approach in response.

All spike count data were reanalyzed using nested hierarchical Poisson generalized linear mixed-effects models (GLMMs), fitted separately for each sex and abstinence duration. Each model included injected current (mean-centered), drug condition, and their interaction as fixed effects, with random intercepts and slopes for injected current at the animal level, and random intercepts for cells nested within animals. Importantly, this reanalysis changed several of our original conclusions. Effects that appeared significant under the conventional cell-level analysis were no longer statistically significant once the hierarchical structure of the data was properly modeled. We report these corrected results transparently throughout the revised manuscript.

However, in males after prolonged abstinence, oxycodone-treated animals showed a higher spike output than controls, with a large effect size. Post-hoc analysis showed only 20% power with current sample (3 saline, 4 oxycodone rats). To reach 80% power, 13 rats per group are needed. We report this as a trend that warrants further study and have revised related sections to reflect this. The data suggest a possible neuroadaptation in males that the study is underpowered to confirm, not a null effect.

In response to this comment, we have updated Figure 5, the Results and Discussion sections, and the Statistics/Methods section to clearly describe the nested hierarchical modeling approach, report corrected statistical values, and acknowledge the power limitation for the male prolonged abstinence group. The figure legend now reports the number of neurons recorded per rat, showing the distribution across animals rather than individual subjects.

(7) The IPSC data, for example in Figure 4, is one of the more novel experiments in the manuscript. However, it is quite challenging to see the difference between males and females, saline and oxycodone, at low stimulation intensities within the graph. Authors should expand this so that reviewers/readers can see those data, especially considering other work suggesting that PVT synaptic input onto select NAc interneurons is disrupted following opioid self-administration. Additional comment: It's also interesting that the IPSC amplitude seems to be maximal at ~2mW of light, whereas ~11 mW is required to evoke maximal EPSC amplitude. It would be interesting to know the authors' thoughts on why this may be.

While visual separation between conditions at low light levels is subtle, we addressed this directly using linear mixed-effects modeling, which evaluates IPSC amplitudes across the full range of stimulation intensities while accounting for repeated measurements from cells nested within animals. This approach provides greater sensitivity than visual inspection alone and avoids over interpretation of noise at individual stimulation levels.

Using this framework, we observed robust main effects of light intensity in both males and females, indicating preserved recruitment of inhibitory synaptic responses as stimulation increased. Importantly, no significant Light × Condition interactions were detected in either sex, indicating that the scaling of IPSC amplitudes with light intensity was not altered by oxycodone exposure.

With respect to the observation that IPSC amplitudes appear to reach near-maximal levels at lower light intensities (~2 mW) compared to EPSCs (~11 mW), we agree that this distinction is intriguing. One possible explanation is that the depend on the recruitment of local interneurons. However, the number of interneurons activated by PVT interneurons is limited and inhibitory responses may reach a plateau at relatively low light intensities once these interneurons are fully recruited.

On the other hand, the increased intensity of photostimulation would result in an increase of monosynaptic EPSC amplitude over a wider range of stimulation (light) intensities, as increased intensity of light would recruit more ChR2-expressing PVT fibers, resulting in larger EPSCs.

(8) There is an inadequate description of what has been done to date on the PVT-NAc projection regarding opioid withdrawal, seeking, disinhibition, and the effects on synaptic physiology therein. For example, a critical paper, Keyes et al., 2020 Neuron, is not cited. Additionally, Paniccia et al., 2024 Neuron is inaccurately cited and insufficiently described. Both manuscripts should be described in some detail within the introduction, and the findings should be accurately contextualized within the broader circuit within the discussion.

In the revised manuscript, we expanded the Discussion to give a more thorough overview of previous research on the PVT-NAc pathway in relation to opioid-related behaviors and synaptic changes. Specifically, we added more detail about Keyes et al., 2020 and Paniccia et al., 2024, clarifying their findings and placing them within the context of the circuit mechanisms studied in our work. We also revised the text to ensure the descriptions of these studies are accurate and that their conclusions are properly related to our findings.

(9) Related to the above, the authors should provide a more comprehensive description of how PVT synapses onto cell-type specific neurons in the NAc which expand beyond MSNs, especially considering that PVT has been shown to influence drug/opioid seeking through the innervation of NAc neurons that are not MSNs. For example, see PMIDs 33947849, 36369508, 28973852, 38141605.

In the revised manuscript, we expanded the Discussion to describe the diversity of PVT projections within the NAc and the potential role of non-MSN neuronal populations in drug-related behaviors. We added discussion on the broader circuit context and other cell types where relevant to the focus on synaptic transmission onto MSNs. Since our experiments specifically examined synaptic physiology in MSNs, we focused the literature discussion on studies most directly related to MSNtargeted PVT inputs and opioid-related behaviors.

Reviewer #3 (Public review):

(10) Additional experiments could strengthen the results and help clarify synaptic mechanisms underpinning behavioral sex differences.

We agree that additional experiments focused on identifying cell-type-specific mechanisms within the PVT-NAcSh circuit would further enhance understanding of the neural substrates behind the observed behavioral sex differences. In the revised manuscript, we have expanded the Discussion to explicitly acknowledge these limitations and clarify the scope of our current study. Specifically, we discuss the possibility that sex-specific adaptations might occur in particular neuronal subpopulations or circuit components that were not resolved in the present experiments. We also mention that future research using cell-type–specific approaches will be necessary to determine if such mechanisms contribute to the increased oxycodone seeking seen in females after prolonged abstinence. We appreciate the reviewer’s suggestions and have incorporated this perspective into the revised manuscript to better contextualize our findings and outline future directions.