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.

  • Article
  • Published:

GABAergic mechanisms regulated by miR-33 encode state-dependent fear


Fear-inducing memories can be state dependent, meaning that they can best be retrieved if the brain states at encoding and retrieval are similar. Restricted access to such memories can present a risk for psychiatric disorders and hamper their treatment. To better understand the mechanisms underlying state-dependent fear, we used a mouse model of contextual fear conditioning. We found that heightened activity of hippocampal extrasynaptic GABAA receptors, believed to impair fear and memory, actually enabled their state-dependent encoding and retrieval. This effect required protein kinase C-βII and was influenced by miR-33, a microRNA that regulates several GABA-related proteins. In the extended hippocampal circuit, extrasynaptic GABAA receptors promoted subcortical, but impaired cortical, activation during memory encoding of context fear. Moreover, suppression of retrosplenial cortical activity, which normally impairs retrieval, had an enhancing effect on the retrieval of state-dependent fear. These mechanisms can serve as treatment targets for managing access to state-dependent memories of stressful experiences.

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

Access options

Buy this article

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

Figure 1: Activation of extrasynaptic GABAA receptors by i.h. injection of gaboxadol induces state-dependent contextual fear.
Figure 2: Expression of gaboxadol-induced state-dependent fear requires PKCβII signaling.
Figure 3: miR-33 is downregulated in response to gaboxadol and modulates its effects on state-dependent fear.
Figure 4: Manipulations of miR-33 alter expression of several GABA-related proteins.
Figure 5: Gaboxadol modulates EGR-1 responses in the hippocampus and its subcortical and cortical projections.
Figure 6: Photomicrographs illustrating the effect of gaboxadol on EGR-1 responses in the hippocampus and its subcortical and cortical projections.
Figure 7: Inactivation of RSC at memory retrieval enhances state-dependent fear.

Similar content being viewed by others


  1. Pompilio, L., Kacelnik, A. & Behmer, S.T. State-dependent learned valuation drives choice in an invertebrate. Science 311, 1613–1615 (2006).

    CAS  PubMed  Google Scholar 

  2. Reus, V.I., Weingartner, H. & Post, R.M. Clinical implications of state-dependent learning. Am. J. Psychiatry 136, 927–931 (1979).

    CAS  PubMed  Google Scholar 

  3. Silberman, E.K., Putnam, F.W., Weingartner, H., Braun, B.G. & Post, R.M. Dissociative states in multiple personality disorder: a quantitative study. Psychiatry Res. 15, 253–260 (1985).

    CAS  PubMed  Google Scholar 

  4. Spiegel, D., Hunt, T. & Dondershine, H.E. Dissociation and hypnotizability in posttraumatic stress disorder. Am. J. Psychiatry 145, 301–305 (1988).

    CAS  PubMed  Google Scholar 

  5. Overton, D.A. Historical context of state-dependent learning and discriminative drug effects. Behav. Pharmacol. 2, 253–264 (1991).

    PubMed  Google Scholar 

  6. Piri, M., Rostampour, M., Nasehi, M. & Zarrindast, M.R. Blockade of the dorsal hippocampal dopamine D1 receptors inhibits the scopolamine-induced state-dependent learning in rats. Neuroscience 252, 460–467 (2013).

    CAS  PubMed  Google Scholar 

  7. Möhler, H. Molecular regulation of cognitive functions and developmental plasticity: impact of GABAA receptors. J. Neurochem. 102, 1–12 (2007).

    PubMed  Google Scholar 

  8. Stein, L. & Berger, B.D. Paradoxical fear-increasing effects of tranquilizers: evidence of repression of memory in the rat. Science 166, 253–256 (1969).

    CAS  PubMed  Google Scholar 

  9. Jacob, T.C., Moss, S.J. & Jurd, R. GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat. Rev. Neurosci. 9, 331–343 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Mortensen, M., Patel, B. & Smart, T.G. GABA potency at GABA(A) receptors found in synaptic and extrasynaptic zones. Front. Cell. Neurosci. 6, 1 (2011).

    PubMed  Google Scholar 

  11. Chandra, D. et al. GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol. Proc. Natl. Acad. Sci. USA 103, 15230–15235 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Blanchard, R.J. & Blanchard, D.C. Crouching as an index of fear. J. Comp. Physiol. Psychol. 67, 370–375 (1969).

    CAS  PubMed  Google Scholar 

  13. Kim, J.J. & Fanselow, M.S. Modality-specific retrograde amnesia of fear. Science 256, 675–677 (1992).

    CAS  PubMed  Google Scholar 

  14. Phillips, R.G. & LeDoux, J.E. Differential contribution of amygdala and hippocampus to cued and contextual fear conditioning. Behav. Neurosci. 106, 274–285 (1992).

    CAS  PubMed  Google Scholar 

  15. Song, M. & Messing, R.O. Protein kinase C regulation of GABAA receptors. Cell. Mol. Life Sci. 62, 119–127 (2005).

    CAS  PubMed  Google Scholar 

  16. Sathyan, P., Golden, H.B. & Miranda, R.C. Competing interactions between micro-RNAs determine neural progenitor survival and proliferation after ethanol exposure: evidence from an ex vivo model of the fetal cerebral cortical neuroepithelium. J. Neurosci. 27, 8546–8557 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Sengupta, J.N. et al. MicroRNA-mediated GABAAalpha-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats. Pain 154, 59–70 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Zhao, C. et al. Computational prediction of MicroRNAs targeting GABA receptors and experimental verification of miR-181, miR-216 and miR-203 targets in GABA-A receptor. BMC Res. Notes 5, 91 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Barmack, N.H., Qian, Z. & Yakhnitsa, V. Long-term climbing fiber activity induces transcription of microRNAs in cerebellar Purkinje cells. Philos. Trans. R. Soc. Lond. B Biol. Sci. 369, 20130508 (2014).

    PubMed  PubMed Central  Google Scholar 

  20. Rayner, K.J. et al. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 328, 1570–1573 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Medrihan, L., Ferrea, E., Greco, B., Baldelli, P. & Benfenati, F. Asynchronous GABA release is a key determinant of tonic inhibition and controls neuronal excitability: a study in the Synapsin II−/− mouse. Cereb. Cortex published online, doi:10.1093/cercor/bhu141 (24 June 2014).

  22. Orsini, C.A., Kim, J.H., Knapska, E. & Maren, S. Hippocampal and prefrontal projections to the basal amygdala mediate contextual regulation of fear after extinction. J. Neurosci. 31, 17269–17277 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Radulovic, J., Kammermeier, J. & Spiess, J. Relationship between fos production and classical fear conditioning: effects of novelty, latent inhibition, and unconditioned stimulus preexposure. J. Neurosci. 18, 7452–7461 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Bozon, B., Davis, S. & Laroche, S. A requirement for the immediate early gene zif268 in reconsolidation of recognition memory after retrieval. Neuron 40, 695–701 (2003).

    CAS  PubMed  Google Scholar 

  25. Xu, W. & Sudhof, T.C. A neural circuit for memory specificity and generalization. Science 339, 1290–1295 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Girden, E. & Culler, E. Conditioned responses in curarized striate muscle in dogs. J. Comp. Psychol. 23, 261–274 (1937).

    Google Scholar 

  27. Corcoran, K.A. et al. NMDA receptors in retrosplenial cortex are necessary for retrieval of recent and remote context fear memory. J. Neurosci. 31, 11655–11659 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Goodwin, D.W., Powell, B., Bremer, D., Hoine, H. & Stern, J. Alcohol and recall: state-dependent effects in man. Science 163, 1358–1360 (1969).

    CAS  PubMed  Google Scholar 

  29. Bustamante, J.A., Jordan, A., Vila, M., Gonzalez, A. & Insua, A. State dependent learning in humans. Physiol. Behav. 5, 793–796 (1970).

    CAS  PubMed  Google Scholar 

  30. Weafer, J., Gallo, D.A. & de Wit, H. Amphetamine fails to alter cued recollection of emotional images: study of encoding, retrieval and state-dependency. PLoS ONE 9, e90423 (2014).

    PubMed  PubMed Central  Google Scholar 

  31. Koek, W. Drug-induced state-dependent learning: review of an operant procedure in rats. Behav. Pharmacol. 22, 430–440 (2011).

    PubMed  Google Scholar 

  32. Colpaert, F.C., Koek, W. & Bruins Slot, L.A. Evidence that mnesic states govern normal and disordered memory. Behav. Pharmacol. 12, 575–589 (2001).

    CAS  PubMed  Google Scholar 

  33. Maren, S., Aharonov, G., Stote, D.L. & Fanselow, M.S. N-methyl-D-aspartate receptors in the basolateral amygdala are required for both acquisition and expression of conditional fear in rats. Behav. Neurosci. 110, 1365–1374 (1996).

    CAS  PubMed  Google Scholar 

  34. Anagnostaras, S.G., Maren, S. & Fanselow, M.S. Scopolamine selectively disrupts the acquisition of contextual fear conditioning in rats. Neurobiol. Learn. Mem. 64, 191–194 (1995).

    CAS  PubMed  Google Scholar 

  35. Davis, M. Diazepam and flurazepam: effects on conditioned fear as measured with the potentiated startle paradigm. Psychopharmacology (Berl.) 62, 1–7 (1979).

    CAS  Google Scholar 

  36. Gao, C. et al. IQGAP1 regulates NR2A signaling, spine density and cognitive processes. J. Neurosci. 31, 8533–8542 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Impey, S. et al. Stimulation of cAMP response element (CRE)-mediated transcription during contextual learning. Nat. Neurosci. 1, 595–601 (1998).

    CAS  PubMed  Google Scholar 

  38. Abel, T. et al. Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampus-based long-term memory. Cell 88, 615–626 (1997).

    CAS  PubMed  Google Scholar 

  39. Merchan, F., Boualem, A., Crespi, M. & Frugier, F. Plant polycistronic precursors containing non-homologous microRNAs target transcripts encoding functionally related proteins. Genome Biol. 10, R136 (2009).

    PubMed  PubMed Central  Google Scholar 

  40. Lin, Q. et al. The brain-specific microRNA miR-128b regulates the formation of fear-extinction memory. Nat. Neurosci. 14, 1115–1117 (2011).

    CAS  PubMed  Google Scholar 

  41. Griggs, E.M., Young, E.J., Rumbaugh, G. & Miller, C.A. MicroRNA-182 regulates amygdala-dependent memory formation. J. Neurosci. 33, 1734–1740 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Brickley, S.G. & Mody, I. Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease. Neuron 73, 23–34 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Moreau, M.P., Bruse, S.E., David-Rus, R., Buyske, S. & Brzustowicz, L.M. Altered microRNA expression profiles in postmortem brain samples from individuals with schizophrenia and bipolar disorder. Biol. Psychiatry 69, 188–193 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Banigan, M.G. et al. Differential expression of exosomal microRNAs in prefrontal cortices of schizophrenia and bipolar disorder patients. PLoS ONE 8, e48814 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Penzes, P., Buonanno, A., Passafaro, M., Sala, C. & Sweet, R.A. Developmental vulnerability of synapses and circuits associated with neuropsychiatric disorders. J. Neurochem. 126, 165–182 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Milenkovic, I. et al. The parvalbumin-positive interneurons in the mouse dentate gyrus express GABAA receptor subunits alpha1, beta2, and delta along their extrasynaptic cell membrane. Neuroscience 254, 80–96 (2013).

    CAS  PubMed  Google Scholar 

  47. Kessler, R.C., Chiu, W.T., Demler, O., Merikangas, K.R. & Walters, E.E. Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication. Arch. Gen. Psychiatry 62, 617–627 (2005).

    PubMed  PubMed Central  Google Scholar 

  48. Franklin, K. & Paxinos, G. The Mouse Brain in Stereotaxic Coordinates (Elsevier, 2004).

  49. Huh, K.H. et al. Hippocampal Erk mechanisms linking prediction error to fear extinction: roles of shock expectancy and contextual aversive valence. Learn. Mem. 16, 273–278 (2009).

    PubMed  PubMed Central  Google Scholar 

  50. Ørom, U.A. & Lund, A.H. Isolation of microRNA targets using biotinylated synthetic microRNAs. Methods 43, 162–165 (2007).

    PubMed  Google Scholar 

  51. Lal, A. et al. Capture of microRNA-bound mRNAs identifies the tumor suppressor miR-34a as a regulator of growth factor signaling. PLoS Genet. 7, e1002363 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Guzmán, Y.F. et al. Fear-enhancing effects of septal oxytocin receptors. Nat. Neurosci. 16, 1185–1187 (2013).

    PubMed  PubMed Central  Google Scholar 

  53. Yamawaki, N., Borges, K., Suter, B.A., Harris, K.D. & Shepherd, G.M. A genuine layer 4 in motor cortex with prototypical synaptic circuit connectivity. Elife 3, e05422 (2014).

    PubMed  PubMed Central  Google Scholar 

  54. Suter, B.A. et al. Ephus: multipurpose data acquisition software for neuroscience experiments. Front. Neural Circuits 4, 100 (2010).

    PubMed  PubMed Central  Google Scholar 

Download references


We thank W. Xu and T.C. Sudhof (Stanford University) for providing us with the SynaptoTag viral vector, C. Fernandez-Hernando (Yale School of Medicine) for providing pMIRNA1/pCDH plasmids encoding miR-33 and scrambled miRNA, and the Genomic Core (Northwestern University) for generating lentiviral vectors containing miR-33 and scrambled constructs. We also thank B. Frick and F. Kassam for their help with the behavioral experiments. This work was supported by US National Institutes of Health grants NIH/NIMH MH078064 (J.R.), NIH/NINDS NS061963 and NS087479 (G.M.G.S.), and a Ken and Ruth Davee Award for Innovative Investigations in Mood Disorders, (J.R. and V.J.).

Author information

Authors and Affiliations



V.J. and J.R. designed the experiments, analyzed the data and wrote the manuscript. V.J. performed the experiments. K.A.C. and K.L. performed some of the behavioral and biochemical experiments. H.J.C. and A.L.G. helped with the biochemical experiments. N.Y. and G.M.G.S. performed the electrophysiological recordings.

Corresponding author

Correspondence to Jelena Radulovic.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Gaboxadol does not affect tone-dependent fear conditioning after i.h. injection.

(a) Gaboxadol (0.1-0.5 µg/hippocampus) did not affect locomotor activity to context or tone at training or activity burst to the footshock (n = 8 mice per group; context 1: F3,22 = 0.814, P = 0.499; tone: F3,22 = 0.431, P = 0.733; shock: F3,22 = 1.109, P = 0.366). (b) In contrast to reduced freezing in context 1, freezing to tone and contextual generalization were unchanged in response to gaboxadol (n = 8 mice per group; context 1: F3,22 = 3.742, P < 0.05; context 2: F3,22 = 0.947, P = 0.434; tone: F3,22 = 0.426, P = 0.737). *P < 0.01 vs vehicle.

Supplementary Figure 2 Scopolamine does not induce state-dependent contextual fear conditioning.

(a) Treatment schedule with scopolamine (25 µg/hippocampus). (b) Scopolamine significantly impaired freezing in all groups including the S-S group, indicating lack of state-dependent contextual fear at doses that impair fear conditioning and memory retrieval (F3,28 = 8.810, P < 0.001). (c) Freezing in the V-S group recovered when mice were tested off drug, whereas the S-V and S-S groups maintained the freezing deficits F3,28 = 3.444, P < 0.05. *P < 0.01 vs V-V group (n = 8 mice/group).

Supplementary Figure 3 Full length images of immunoblots showing increased phosphorylation of PKC βII.

(a) Phosphorylation of PKC isoforms 1 and 24 hrs after fear conditioning. (b) Phosphorylation of PKC βII immeadiately after testing of V-G nd G-G mice.

Supplementary Figure 4 Differential expression of microRNAs that target GABA-related proteins during fear conditioning.

(a) Microarrays of microRNAs differentially expressed in hippocampi of mice after fear conditioning (F) that show > 50% change when compared to control hippocampi from naïve (N) mice. Of nineteen identified microRNAs, fourteen have predicted targets within GABAA receptors, and five of them (marked red) have four or more predicted GABAR targets. (b) Conserved miR-33 targets in GABA-related proteins.

Supplementary Figure 5 Validation of the miR-33 manipulation.

(a) The plasmid construct carrying miR-33 indicates lack of viral toxicity as revealed by viable and healthy neuroblastoma cells infected with LV-SCR or LV-miR-33. (b) qPCR analysis of relative miR-33 level in cells infected with LV-miR-33 vs LV-SCR (t6 = -3.508, P < 0.05, t-test). (c) In vivo validation of the miR-33 overexpression in the mouse hippocampus obtained from mice tested as described in Fig. 3b (n = 5 hippocampi/group; t9=10.297, P < 0.01). (d) Dose-dependent inhibition of miR-33 after i.h. injection of miR-33 LNA when compared to miR-S-LNA (F4,14 = 48.368, P < 0.001, one-way ANOVA). miR-124 levels were determined as a specificity control. (e) and (f) In vivo validation of the miR-33 down-regulation in the mouse hippocampus obtained from mice tested as described in Fig. 3c,d (n = 4 hippocampi/group; t7=8.715, P < 0.01 and n = 4 hippocampi/group; t7=9.12, P < 0.001). *P < 0.05, **P < 0.01, *** P < 0.001 vs corresponding controls.

Supplementary Figure 6 Virus spread and schematic representation of the treatment schedule for the miR-33 experiments.

(a) Spread of lentiviruses along the dorso-ventral hippocampus as revealed by immunohistochemistry with anti-GFP antibodies. (b) Treatment schedule for miR-33 overexpression with LV-miR-33. (c) Treatment schedule for miR-33 inhibition with miR-33-LNA.

Supplementary Figure 7 miR-33 manipulations affects the level of GABA-related proteins but not the level of NMDAR and protein kinases typically required for fear conditioning.

(a) Immunoblots showing the effect of miR-33 overexpression and (b) miR-33 inhibition on the levels of GABRA4, GABRB2, KCC2 and Syn2a,b (quantification shown in Figs. 4b,c,e). (c) miR-33 overexpression did not affect the level of the main NMDAR subunits NR1, NR2A, or NR2B (5 samples/protein/treatment; F2,24 = 2.116, P = 0.115) or (d) protein kinases cAMP-dependent protein kinase (PKA), calcium and calmodulin-regulated kinase II (CaMKII), or extracellular signal-regulated kinases 1/2 (Erk-1/2) (5 samples/protein/treatment; F2,24 = 0.31, P = 0.861).

Supplementary Figure 8 Proteomic analyses of hippocampal protein samples after miR-S-LNA or miR-33-LNA treatment.

(a) 2D Gel Difference Image of averaged Sample A (vehicle-injected group) vs averaged Sample B (gaboxadol-injected group). Polypeptide spots increased in Sample A vs Sample B are outlined in Blue, while spots decreased in Sample A vs Sample B are outlined in Red. (b) The spot later identified as synapsin-2. (c) Identification of differentially produced proteins by mass spectrophotometry.

Supplementary Figure 9 Coordinates for infusions and immediate early gene response quantification.

(a) Hippocampal coordinates used for infusion of drugs, viruses, and microRNAs (left), and an example of cannula placement (right). (b) Higher magnification showing cannula traces in the CA1 subfield of mice injected with SynaptoTag followed by gaboxadol in images showing synapsin (green) and mCherry (red) (left) or EGR-1 (blue, right). (c) Coordinates used for quantification of EGR-1 and cFos immunostaining.

Supplementary Figure 10 cFos responses after fear conditioning with gaboxadol.

Individual photomicrographs of average cFos (pseudocolored green) immunostaining in different groups and brain areas. Significant changes were found in the lateral septum (F2,8 = 5.232, P < 0.05) *P < 0.05, **P < 0.01, when compared to the V group.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 1571 kb)

Supplementary Methods Checklist

(PDF 455 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jovasevic, V., Corcoran, K., Leaderbrand, K. et al. GABAergic mechanisms regulated by miR-33 encode state-dependent fear. Nat Neurosci 18, 1265–1271 (2015).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing