NPAS4 in the medial prefrontal cortex mediates chronic social defeat stress-induced anhedonia-like behavior and reductions in excitatory synapses
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This is a very interesting manuscript that will be of interest to the field of stress neurobiology and neuropsychiatry. Claims about the interactions between stress and medial prefrontal cortex NPAS4 on anhedonia and motivation remain to be firmly established, yet clear evidence is provided for NPAS4 function on medial prefrontal cortex pyramidal neuron dendritic morphology and gene expression.
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Abstract
Chronic stress can produce reward system deficits (i.e., anhedonia) and other common symptoms associated with depressive disorders, as well as neural circuit hypofunction in the medial prefrontal cortex (mPFC). However, the molecular mechanisms by which chronic stress promotes depressive-like behavior and hypofrontality remain unclear. We show here that the neuronal activity-regulated transcription factor, NPAS4, in the mPFC is regulated by chronic social defeat stress (CSDS), and it is required in this brain region for CSDS-induced changes in sucrose preference and natural reward motivation in the mice. Interestingly, NPAS4 is not required for CSDS-induced social avoidance or anxiety-like behavior. We also find that mPFC NPAS4 is required for CSDS-induced reductions in pyramidal neuron dendritic spine density, excitatory synaptic transmission, and presynaptic function, revealing a relationship between perturbation in excitatory synaptic transmission and the expression of anhedonia-like behavior in the mice. Finally, analysis of the mice mPFC tissues revealed that NPAS4 regulates the expression of numerous genes linked to glutamatergic synapses and ribosomal function, the expression of upregulated genes in CSDS-susceptible animals, and differentially expressed genes in postmortem human brains of patients with common neuropsychiatric disorders, including depression. Together, our findings position NPAS4 as a key mediator of chronic stress-induced hypofrontal states and anhedonia-like behavior.
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Author Response
Reviewer #1 (Public Review):
The current study uses microbiology, biochemistry, microscopy, and viral vectors to establish a role for prefrontal cortex expression of the immediate early gene NPAS4 in sucrose preference and dendritic spine morphology in the mouse social defeat stress model. The experimental designs are appropriate and the hypotheses addressed are interesting. The paper is generally very well-written and the figures are clear. Most of the statistical analyses are appropriate, and they are reported in clear and useful tables. Thus, the general potential for the studies is quite high. The authors conclusively show that NPAS4 is induced in mPFC in response to social defeat stress and that NPAS4 is important for stress-induced changes in mPFC dendritic spine number. However, some of the key data regarding …
Author Response
Reviewer #1 (Public Review):
The current study uses microbiology, biochemistry, microscopy, and viral vectors to establish a role for prefrontal cortex expression of the immediate early gene NPAS4 in sucrose preference and dendritic spine morphology in the mouse social defeat stress model. The experimental designs are appropriate and the hypotheses addressed are interesting. The paper is generally very well-written and the figures are clear. Most of the statistical analyses are appropriate, and they are reported in clear and useful tables. Thus, the general potential for the studies is quite high. The authors conclusively show that NPAS4 is induced in mPFC in response to social defeat stress and that NPAS4 is important for stress-induced changes in mPFC dendritic spine number. However, some of the key data regarding reward motivation are difficult to properly interpret and do not convincingly demonstrate a behavioral result of NPAS4 knockdown in mPFC. Moreover, the spine morphology and sequencing analyses lack depth. Most importantly, although the authors explore the effects of reducing NPAS4 expression in mPFC, they do not explore the effects of increasing NPAS4 expression or function, and thus the studies seem incomplete and cannot be fully interpreted.
We appreciate the reviewer's overall positive feedback on our study and the constructive comments to improve the manuscript. In the revised document, we have addressed the key concerns about NPAS4’s function on motivated behavior by providing the new data by which NPAS4 limits natural reward motivation in the CSDS-susceptible group (Figure 3C-D). We encountered the major challenge that animals that sustained injuries during CSDS had to be removed from the study resulting in relatively few susceptible mice. Other factors likely contributed to the low proportion of susceptible mice, including the biological sex of the investigator (Georgiou et al., Nature Neuroscience, 2022). For the gene expression analysis, we provided comparative analysis of our RNA-seq data with published NPAS4 ChIP-seq data to demonstrate genome-wide NPAS4 association, suggesting potential direct NPAS4 target genes. Furthermore, to extend the structural synapse data, we now provide new electrophysiology data (Figure 4C-H). These new data demonstrate that NPAS4 is required for the CSDS-induced reduction of mEPSC frequency. Using new single-nuclei RNA-seq data from adult mPFC tissues, we observe that NPAS4 is expressed predominantly (~93%) in excitatory neuron clusters, but is also expressed in multiple interneuron populations (~7%). Since our NPAS4 knockdown strategy is not cell type-specific, we have revised the discussion to reflect the possibility that some of the NPAS4-dependent CSDS effects on structural and functional glutamatergic synapses and anhedonia-like behaviors could be due, at least in part, to NPAS4 function in one or more classes of GABAergic interneurons. We have discussed these limitations of interpretation, and the need for future cell type-specific approaches, in the revised manuscript.
Reviewer #2 (Public Review):
The authors investigate whether neuronal activity-regulated transcription factor 4 (NPAS4) in the medial prefrontal cortex (mPFC) is involved in stress-induced effects on neuronal spine synapse density (as a proxy for synaptic activity) and reward behaviors. A major strength of the manuscript is that NPAS4 is shown to be necessary for stress-induced reward deficits and pyramidal neuron spine density. In addition, whole transcriptome analysis of NPAS4 target genes identify a number of genes previously found to be regulated in the postmortem brain of humans with MDD, providing translational relevance to these studies. A weakness is that studies were only performed in male mice so its unclear how generalizable these effects are to females. Despite this, the work will likely impact the field of neuropsychiatry by providing novel information about the molecular and cellular mechanisms in mPFC responsible for stressinduced effects on spines synapses and reward behaviors.
We would like to thank the reviewer for the positive comments, including comparison of our NPAS4-dependent PFC genes with published data from postmortem brains of human’s diagnosed with MDD. We agree with the reviewer that assessing the role of NPAS4 in CSDS or similar chronic stress paradigm in females will be an important future direction for our work, and we acknowledge this limitation of our study in the revised manuscript.
Reviewer #3 (Public Review):
Hughes et al. report a role for the transcription factor NPAS4 in mediating chronic stressinduced reward-related behavioral changes, but not other depression-like behaviors. The authors find that NPAS4 is transiently upregulated in Camk2a+ PFC neurons following a single bout or repeated social defeat stress, and that knocking down PFC Npas4 prevents anhedonia. Presentation of linked individual data for social interaction/avoidance measures with/without interaction partners (Fig2C, E) is commended - all CSDS papers should show data this way. Npas4 also appears to mediate the known effect of stress on spines in PFC, providing novel mechanistic insight into this phenomenon. Npas4 knockdown altered baseline transcription in PFC, which overlapped with other stress and MDD-associated transcriptional changes and modules. However, stress-induced changes in transcription with knockdown remain unknown. A major drawback is that only male mice were used, although this is discussed to some extent. Results are presented with appropriate context and references to the literature. Conclusions are appropriate.
Additional context: Given NPAS4's role as an immediate early gene, it will be important for future work to elucidate whether IEG knockdown generally dampens transcriptional response to stress/other salient experiences. Nevertheless, the authors do show several pieces of evidence that Npas4 knockdown does not simply make mice less sensitive to stress and/or produce deficits in threat/fear-related learning and memory which is an important piece of this puzzle.
We appreciate the thoughtful and generous comments from the reviewer regarding our display method for social interaction/avoidance data. We agree that a major limitation of our study is the lack of females. Unfortunately, we’ve had limited success with reported adaptations for the use of females in CSDS, and follow-up studies will be critical to assess NPAS4’s mPFC role in chronic stress-induced anhedonia-like behavior. We address this limitation of our current study in the discussion section.
We agree that IEG manipulation might produce profound changes in the stress-dependent transcriptome of the mPFC. Toward this goal, we investigated the gene expression of several candidate NPAS4 target genes at 1-hour after acute social defeat stress, a timepoint of nearpeak protein expression of NPAS4 (Supplemental Figure 4). Although we observed a main effect of Npas4 knockdown, we did not observe an impact of NPAS4 on stress-induced gene expression (Supplemental Figure 4). NPAS4 is a very rapidly and transiently expressed by stress and neural activity, so to determine the impacts of NPAS4 on stress-induced changes in transcription, multiple time points of research will need to be examined. Future studies performing single-cell transcriptomics at various time points following acute or chronic social defeat stress, sucrose SA, and social interaction will be important to address these questions.
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eLife assessment
This is a very interesting manuscript that will be of interest to the field of stress neurobiology and neuropsychiatry. Claims about the interactions between stress and medial prefrontal cortex NPAS4 on anhedonia and motivation remain to be firmly established, yet clear evidence is provided for NPAS4 function on medial prefrontal cortex pyramidal neuron dendritic morphology and gene expression.
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Reviewer #1 (Public Review):
The current study uses microbiology, biochemistry, microscopy, and viral vectors to establish a role for prefrontal cortex expression of the immediate early gene NPAS4 in sucrose preference and dendritic spine morphology in the mouse social defeat stress model. The experimental designs are appropriate and the hypotheses addressed are interesting. The paper is generally very well-written and the figures are clear. Most of the statistical analyses are appropriate, and they are reported in clear and useful tables. Thus, the general potential for the studies is quite high. The authors conclusively show that NPAS4 is induced in mPFC in response to social defeat stress and that NPAS4 is important for stress-induced changes in mPFC dendritic spine number. However, some of the key data regarding reward motivation …
Reviewer #1 (Public Review):
The current study uses microbiology, biochemistry, microscopy, and viral vectors to establish a role for prefrontal cortex expression of the immediate early gene NPAS4 in sucrose preference and dendritic spine morphology in the mouse social defeat stress model. The experimental designs are appropriate and the hypotheses addressed are interesting. The paper is generally very well-written and the figures are clear. Most of the statistical analyses are appropriate, and they are reported in clear and useful tables. Thus, the general potential for the studies is quite high. The authors conclusively show that NPAS4 is induced in mPFC in response to social defeat stress and that NPAS4 is important for stress-induced changes in mPFC dendritic spine number. However, some of the key data regarding reward motivation are difficult to properly interpret and do not convincingly demonstrate a behavioral result of NPAS4 knockdown in mPFC. Moreover, the spine morphology and sequencing analyses lack depth. Most importantly, although the authors explore the effects of reducing NPAS4 expression in mPFC, they do not explore the effects of increasing NPAS4 expression or function, and thus the studies seem incomplete and cannot be fully interpreted.
-
Reviewer #2 (Public Review):
The authors investigate whether neuronal activity-regulated transcription factor 4 (NPAS4) in the medial prefrontal cortex (mPFC) is involved in stress-induced effects on neuronal spine synapse density (as a proxy for synaptic activity) and reward behaviors. A major strength of the manuscript is that NPAS4 is shown to be necessary for stress-induced reward deficits and pyramidal neuron spine density. In addition, whole transcriptome analysis of NPAS4 target genes identify a number of genes previously found to be regulated in the postmortem brain of humans with MDD, providing translational relevance to these studies. A weakness is that studies were only performed in male mice so its unclear how generalizable these effects are to females. Despite this, the work will likely impact the field of neuropsychiatry …
Reviewer #2 (Public Review):
The authors investigate whether neuronal activity-regulated transcription factor 4 (NPAS4) in the medial prefrontal cortex (mPFC) is involved in stress-induced effects on neuronal spine synapse density (as a proxy for synaptic activity) and reward behaviors. A major strength of the manuscript is that NPAS4 is shown to be necessary for stress-induced reward deficits and pyramidal neuron spine density. In addition, whole transcriptome analysis of NPAS4 target genes identify a number of genes previously found to be regulated in the postmortem brain of humans with MDD, providing translational relevance to these studies. A weakness is that studies were only performed in male mice so its unclear how generalizable these effects are to females. Despite this, the work will likely impact the field of neuropsychiatry by providing novel information about the molecular and cellular mechanisms in mPFC responsible for stress-induced effects on spines synapses and reward behaviors.
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Reviewer #3 (Public Review):
Hughes et al. report a role for the transcription factor NPAS4 in mediating chronic stress-induced reward-related behavioral changes, but not other depression-like behaviors. The authors find that NPAS4 is transiently upregulated in Camk2a+ PFC neurons following a single bout or repeated social defeat stress, and that knocking down PFC Npas4 prevents anhedonia. Presentation of linked individual data for social interaction/avoidance measures with/without interaction partners (Fig2C, E) is commended - all CSDS papers should show data this way. Npas4 also appears to mediate the known effect of stress on spines in PFC, providing novel mechanistic insight into this phenomenon. Npas4 knockdown altered baseline transcription in PFC, which overlapped with other stress and MDD-associated transcriptional changes and …
Reviewer #3 (Public Review):
Hughes et al. report a role for the transcription factor NPAS4 in mediating chronic stress-induced reward-related behavioral changes, but not other depression-like behaviors. The authors find that NPAS4 is transiently upregulated in Camk2a+ PFC neurons following a single bout or repeated social defeat stress, and that knocking down PFC Npas4 prevents anhedonia. Presentation of linked individual data for social interaction/avoidance measures with/without interaction partners (Fig2C, E) is commended - all CSDS papers should show data this way. Npas4 also appears to mediate the known effect of stress on spines in PFC, providing novel mechanistic insight into this phenomenon. Npas4 knockdown altered baseline transcription in PFC, which overlapped with other stress and MDD-associated transcriptional changes and modules. However, stress-induced changes in transcription with knockdown remain unknown. A major drawback is that only male mice were used, although this is discussed to some extent. Results are presented with appropriate context and references to the literature. Conclusions are appropriate.
Additional context: Given NPAS4's role as an immediate early gene, it will be important for future work to elucidate whether IEG knockdown generally dampens transcriptional response to stress/other salient experiences. Nevertheless, the authors do show several pieces of evidence that Npas4 knockdown does not simply make mice less sensitive to stress and/or produce deficits in threat/fear-related learning and memory which is an important piece of this puzzle.
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