Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Microglial P2Y12 mediates chronic stress-induced synapse loss in the prefrontal cortex and associated behavioral consequences


Chronic unpredictable stress (CUS) drives microglia-mediated neuronal remodeling and synapse loss in the prefrontal cortex (PFC), contributing to deficits in cognition and behavior. However, it remains unclear what mechanisms guide microglia-neuron interactions in stress. Evidence indicates that neuronal activity-dependent purinergic signaling directs microglial processes and synaptic engagement via P2Y12, a purinergic receptor exclusively expressed by microglia in the brain. Stress alters excitatory neurotransmission in the PFC, thus we aimed to determine if P2Y12 signaling promotes functional changes in microglia in chronic stress. Here we used genetic ablation of P2Y12 (P2ry12–/–) or pharmacological blockade (clopidogrel, ticagrelor) to examine the role of purinergic signaling in stress-induced microglia-neuron interaction. Multiple behavioral, physiological, and cytometric endpoints were analyzed. Deletion of P2Y12 led to a number of fundamental alterations in the PFC, including the heightened microglial number and increased dendritic spine density. Flow cytometry revealed that microglia in P2ry12–/– mice had shifts in surface levels of CX3CR1, CSF1R, and CD11b, suggesting changes in synaptic engagement and phagocytosis in the PFC. In line with this, pharmacological blockade of P2Y12 prevented CUS-induced increases in the proportion of microglia with neuronal inclusions, limited dendritic spine loss in the PFC, and attenuated alterations in stress coping behavior and working memory function. Overall, these findings indicate that microglial P2Y12 is a critical mediator of stress-induced synapse loss in the PFC and subsequent behavioral deficits.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Constitutive ablation of P2Y12 attenuates stress effects on coping behavior and working memory, and induces significant alterations in frontal cortex microglia.
Fig. 2: Loss of P2Y12 increases lysosome markers in the medial prefrontal cortex and prevents dendritic remodeling in chronic stress.
Fig. 3: Pharmacological blockade of microglial P2Y12 attenuates stress effects on behavior and shifts microglial phenotype in the frontal cortex.
Fig. 4: Administration of clopidogrel reduces levels of microglial P2Y12 and prevents stress effects on microglial morphology.
Fig. 5: Antagonism of microglial P2Y12 prevents stress-induced phagocytosis of dendritic elements and subsequent dendritic spine loss in the mPFC.


  1. Thompson SM, Kallarackal AJ, Kvarta MD, Van Dyke AM, LeGates TA, Cai X. An excitatory synapse hypothesis of depression. Trends Neurosci. 2015;38:279–94.

    Article  CAS  Google Scholar 

  2. Duman RS, Aghajanian GK, Sanacora G, Krystal JH. Synaptic plasticity and depression: New insights from stress and rapid-acting antidepressants. Nat Med. 2016;22:238–49.

  3. Holmes SE, Scheinost D, Finnema SJ, Naganawa M, Davis MT, DellaGioia N, et al. Lower synaptic density is associated with depression severity and network alterations. Nature Commun. 2019;10:1529.

    Article  Google Scholar 

  4. Liu T, Lu J, Lukasiewicz K, Pan B, Zuo Y. Stress induces microglia-associated synaptic circuit alterations in the dorsomedial prefrontal cortex. Neurobiol Stress. 2021:100342.

  5. Wohleb ES, Terwilliger R, Duman CH, Duman RS. Stress-induced neuronal CSF1 provokes microglia-mediated neuronal remodeling and depressive-like behavior. Biol Psychiatry. 2018;83:38–49.

    Article  CAS  Google Scholar 

  6. Yuen EY, Liu W, Karatsoreos IN, Feng J, McEwen BS, Yan Z. Acute stress enhances glutamatergic transmission in prefrontal cortex and facilitates working memory. Proc Natl Acad Sci USA. 2009;106:14075–9.

    Article  CAS  Google Scholar 

  7. Miner LAH, Jedema HP, Moore FW, Blakely RD, Grace AA, Sesack SR. Chronic stress increases the plasmalemmal distribution of the norepinephrine transporter and the coexpression of tyrosine hydroxylase in norepinephrine axons in the prefrontal cortex. J Neurosci. 2006;26:1571–8.

    Article  CAS  Google Scholar 

  8. Haynes SE, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan W-B, et al. The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci. 2006;9:1512.

    Article  CAS  Google Scholar 

  9. Dissing-Olesen L, LeDue JM, Rungta RL, Hefendehl JK, Choi HB, MacVicar BA. Activation of neuronal NMDA receptors triggers transient ATP-mediated microglial process outgrowth. J Neurosci. 2014;34:10511.

    Article  Google Scholar 

  10. Sipe GO, Lowery RL, Tremblay M, Kelly EA, Lamantia CE, Majewska AK. Microglial P2Y12 is necessary for synaptic plasticity in mouse visual cortex. Nature. Communications. 2016;7:1–15.

    Google Scholar 

  11. Butovsky O, Jedrychowski MP, Moore CS, Cialic R, Lanser AJ, Gabriely G, et al. Identification of a unique TGF-β–dependent molecular and functional signature in microglia. Nat Neurosci. 2013;17:131–43.

    Article  Google Scholar 

  12. Eyo UB, Peng J, Swiatkowski P, Mukherjee A, Bispo A, Wu L-JJ. Neuronal hyperactivity recruits microglial processes via neuronal NMDA receptors and microglial P2Y12 receptors after status epilepticus. J Neurosci. 2014;34:10528–40.

    Article  Google Scholar 

  13. Woodburn SC, Bollinger JL, Wohleb ES. Synaptic and behavioral effects of chronic stress are linked to dynamic and sex-specific changes in microglia function and astrocyte dystrophy. Neurobiol Stress. 2021;14:100312.

  14. Horchar MJ, Wohleb ES. Glucocorticoid receptor antagonism prevents microglia-mediated neuronal remodeling and behavioral despair following chronic unpredictable stress. Brain, Behavior, Immunity. 2019. 2019.

  15. Tozaki-Saitoh H, Tsuda M, Miyata H, Ueda K, Kohsaka S, Inoue K. P2Y12 receptors in spinal microglia are required for neuropathic pain after peripheral nerve injury. J Neurosci. 2008;28:4949–56.

    Article  CAS  Google Scholar 

  16. Yuen EY, Wei J, Liu W, Zhong P, Li X, Yan Z. Repeated stress causes cognitive impairment by suppressing glutamate receptor expression and function in prefrontal cortex. Neuron. 2012;73:962–77.

    Article  CAS  Google Scholar 

  17. Warden MR, Selimbeyoglu A, Mirzabekov JJ, Lo M, Thompson KR, Kim SY, et al. A prefrontal cortex–brainstem neuronal projection that controls response to behavioural challenge. Nature. 2012;492:428–32. 7429. 2012;492

    Article  CAS  Google Scholar 

  18. Barker GRI, Bird F, Alexander V, Warburton EC. Recognition memory for objects, place, and temporal order: A disconnection analysis of the role of the medial prefrontal cortex and perirhinal cortex. J Neurosci. 2007. 2007.

  19. Porrero C, Rubio-Garrido P, Avendaño C, Clascá F. Mapping of fluorescent protein-expressing neurons and axon pathways in adult and developing Thy1-eYFP-H transgenic mice. Brain Res. 2010;1345:59–72.

    Article  CAS  Google Scholar 

  20. Weinhard L, di Bartolomei G, Bolasco G, Machado P, Schieber NL, Neniskyte U, et al. Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction. Nat Commun. 2018;9:1228.

    Article  Google Scholar 

  21. Wei J, Carroll RJ, Harden KK, Wu G. Comparisons of treatment means when factors do not interact in two-factorial studies. Amino Acids. 2012;42:2031–5.

    Article  CAS  Google Scholar 

  22. Weissgerber TL, Garcia-Valencia O, Garovic VD, Milic NM, Winham SJ. Why we need to report more than ‘Data were Analyzed by t-tests or ANOVA’. ELife. 2018;7:e36163.

    Article  Google Scholar 

  23. Ruxton GD, Beauchamp G. Time for some a priori thinking about post hoc testing. Behav Ecol. 2008;19:690–3.

    Article  Google Scholar 

  24. Alger BE. Neuroscience needs to test both statistical and scientific hypotheses. J Neurosci. 2022;42:8432–8.

    Article  CAS  Google Scholar 

  25. Burns JC, Cotleur B, Walther DM, Bajrami B, Rubino SJ, Wei R, et al. Differential accumulation of storage bodies with aging defines discrete subsets of microglia in the healthy brain. ELife. 2020;9:1–71.

    Article  Google Scholar 

  26. Bollinger JL, Horchar MJ, Wohleb ES. Diazepam limits microglia-mediated neuronal remodeling in the prefrontal cortex and associated behavioral consequences following chronic unpredictable stress. Neuropsychopharmacology. 2020;45:1766–76.

    Article  CAS  Google Scholar 

  27. Hickman SE, Kingery ND, Ohsumi TK, Borowsky ML, Wang LC, Means TK, et al. The microglial sensome revealed by direct RNA sequencing. Nat Neurosci. 2013;16:1896–905.

    Article  CAS  Google Scholar 

  28. Eyo UB, Mo M, Yi M-H, Murugan M, Liu J, Yarlagadda R, et al. P2Y12R-dependent translocation mechanisms gate the changing microglial landscape. Cell Rep. 2018;23:959–66.

    Article  CAS  Google Scholar 

  29. Mo M, Eyo UB, Xie M, Peng J, Bosco DB, Umpierre AD, et al. Microglial P2Y12 receptor regulates seizure-induced neurogenesis and immature neuronal projections. J. Neurosci. 2019;39:9453–64.

    Article  CAS  Google Scholar 

  30. Schafer DP, Lehrman EK, Kautzman AG, Koyama R, Mardinly AR, Yamasaki R, et al. Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner. Neuron. 2012;74:691–705.

    Article  CAS  Google Scholar 

  31. Paolicelli RC, Bolasco G, Pagani F, Maggi L, Scianni M, Panzanelli P, et al. Synaptic pruning by microglia is necessary for normal brain development. Science. 2011;333:1456–8.

    Article  CAS  Google Scholar 

  32. Cardona AE, Pioro EP, Sasse ME, Kostenko V, Cardona SM, Dijkstra IM, et al. Control of microglial neurotoxicity by the fractalkine receptor. Nat Neurosci. 2006;9:917–24.

    Article  CAS  Google Scholar 

  33. Lowery RL, Mendes MS, Sanders BT, Murphy AJ, Whitelaw BS, Lamantia CE, et al. Loss of P2Y12 has behavioral effects in the adult mouse. Int J Mol Sci. 2021;22:1868.

    Article  CAS  Google Scholar 

  34. Hamilton PJ, Chen EY, Tolstikov V, Peña CJ, Picone JA, Shah P, et al. Chronic stress and antidepressant treatment alter purine metabolism and beta oxidation within mouse brain and serum. Sci Rep. 2020;10:18134.

  35. Yue N, Huang H, Zhu X, Han Q, Wang Y, Li B, et al. Activation of P2X7 receptor and NLRP3 inflammasome assembly in hippocampal glial cells mediates chronic stress-induced depressive-like behaviors. J Neuroinflammation. 2017;14:102.

    Article  Google Scholar 

  36. Badimon A, Strasburger HJ, Ayata P, Chen X, Nair A, Ikegami A, et al. Negative feedback control of neuronal activity by microglia. Nature. 2020;586:417–23.

    Article  CAS  Google Scholar 

  37. Lou N, Takano T, Pei Y, Xavier AL, Goldman SA, Nedergaard M. Purinergic receptor P2RY12-dependent microglial closure of the injured blood–brain barrier. Proc Natl Acad Sci. 2016;113:1074–9.

    Article  CAS  Google Scholar 

  38. Wohleb ES, Terwilliger R, Duman CH, Duman RS. Stress-induced neuronal colony stimulating factor 1 provokes microglia-mediated neuronal remodeling and depressive-like behavior. Biol Psychiatry. 2018;83:38–49.

    Article  CAS  Google Scholar 

  39. El Hajj H, Savage JC, Bisht K, Parent M, Vallières L, Rivest S, et al. Ultrastructural evidence of microglial heterogeneity in Alzheimer’s disease amyloid pathology. J Neuroinflammation. 2019;16:87.

    Article  Google Scholar 

  40. Peng J, Liu Y, Umpierre AD, Xie M, Tian DS, Richardson JR, et al. Microglial P2Y12 receptor regulates ventral hippocampal CA1 neuronal excitability and innate fear in mice. Mol Brain. 2019;12:71.

  41. Yu T, Zhang X, Shi H, Tian J, Sun L, Hu X, et al. P2Y12 regulates microglia activation and excitatory synaptic transmission in spinal lamina II neurons during neuropathic pain in rodents. Cell Death Dis. 2019;10:1–16. 10:3. 2019

    Article  Google Scholar 

  42. Bollinger JL, Burns CMB, Wellman CL. Differential effects of stress on microglial cell activation in male and female medial prefrontal cortex. Brain Behav Immunity. 2016;52:88–97.

    Article  CAS  Google Scholar 

  43. Bollinger JL, Salinas I, Fender E, Sengelaub DR, Wellman CL. Gonadal hormones differentially regulate sex-specific stress effects on glia in the medial prefrontal cortex. J Neuroendocrinol. 2019. 2019.

  44. Gaspar R, Soares-Cunha C, Domingues AV, Coimbra B, Baptista FI, Pinto L, et al. Resilience to stress and sex-specific remodeling of microglia and neuronal morphology in a rat model of anxiety and anhedonia. Neurobiol Stress. 2021;14:100302.

  45. Bolton JL, Short AK, Othy S, Kooiker CL, Shao M, Gunn BG, et al. Early stress-induced impaired microglial pruning of excitatory synapses on immature CRH-expressing neurons provokes aberrant adult stress responses. Cell Rep. 2022;38:110600.

    Article  CAS  Google Scholar 

  46. Tsyglakova M, Huskey AM, Hurst EH, Telep NM, Wilding MC, Babington ME, et al. Sex and region-specific effects of variable stress on microglia morphology. Brain, Behav Immunity - Health. 2021;18:100378.

    Article  Google Scholar 

  47. Bollinger JL, Collins KE, Patel R, Wellman CL. Behavioral stress alters corticolimbic microglia in a sex- and brain region-specific manner. PLOS ONE. 2017;12:e0187631.

    Article  Google Scholar 

  48. Caetano L, Pinheiro H, Patrício P, Mateus-Pinheiro A, Alves ND, Coimbra B, et al. Adenosine A2A receptor regulation of microglia morphological remodeling-gender bias in physiology and in a model of chronic anxiety. Mol Psychiatry. 2017;22:1035–43.

    Article  CAS  Google Scholar 

  49. Seney ML, Huo Z, Cahill K, French L, Puralewski R, Zhang J, et al. Opposite Molecular Signatures of Depression in Men and Women. Biol Psychiatry. 2018;84:18–27.

    Article  CAS  Google Scholar 

  50. Bollinger JL. Uncovering microglial pathways driving sex-specific neurobiological effects in stress and depression. Brain, Behav Immunity - Health. 2021;16:100320.

    Article  CAS  Google Scholar 

  51. Böttcher C, Fernández-Zapata C, Snijders GJL, Schlickeiser S, Sneeboer MAM, Kunkel D, et al. Single-cell mass cytometry of microglia in major depressive disorder reveals a non-inflammatory phenotype with increased homeostatic marker expression. Transl Psychiatry. 2020;10:1–11.

    Article  Google Scholar 

  52. Snijders GJLJ, Sneeboer MAM, Fernández-Andreu A, Udine E, Boks MP, Ormel PR, et al. Distinct non-inflammatory signature of microglia in post-mortem brain tissue of patients with major depressive disorder. Mol Psychiatry. 2020; 26:3336–49.

  53. Woodburn SC, Bollinger JL, Wohleb ES. The semantics of microglia activation: neuroinflammation, homeostasis, and stress. J Neuroinflammation. 2021;18:1–16. 1. 2021;18

    Article  Google Scholar 

Download references


The authors would like to thank Dr. Ania Majewska for kindly donating P2ry12–/– mice, and Dr. Grayson Sipe for advice regarding administration of clopidogrel and ticagrelor. The authors would also like to thank Dr. Lauren Vollmer for feedback on this manuscript. Additional support was provided by the Statistics Consulting Center at the University of Cincinnati. This work was supported by the National Institute of Mental Health (F32MH123051, JLB; R01MH123545, ESW) and the University of Cincinnati.

Author information

Authors and Affiliations



Study conception and design: JLB, ESW. Acquisition of data: JLB, DTD, JKF, ILR, SCW. Analysis and interpretation of data: JLB, ESW. Drafting of paper: JLB, ESW.

Corresponding author

Correspondence to Eric S. Wohleb.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bollinger, J.L., Dadosky, D.T., Flurer, J.K. et al. Microglial P2Y12 mediates chronic stress-induced synapse loss in the prefrontal cortex and associated behavioral consequences. Neuropsychopharmacol. (2022).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI:


Quick links