Perineuronal net formation during the critical period for neuronal maturation in the hypothalamic arcuate nucleus


In leptin-deficient ob/ob mice, obesity and diabetes are associated with abnormal development of neurocircuits in the hypothalamic arcuate nucleus (ARC)1, a critical brain area for energy and glucose homoeostasis2,3. Because this developmental defect can be remedied by systemic leptin administration, but only if given before postnatal day 28, a critical period for leptin-dependent development of ARC neurocircuits has been proposed4. In other brain areas, critical-period closure coincides with the appearance of perineuronal nets (PNNs), extracellular matrix specializations that restrict the plasticity of neurons that they enmesh5. Here we report that in humans and rodents, subsets of neurons in the mediobasal aspect of the ARC are enmeshed in PNN-like structures. In mice, these neurons are densely packed into a continuous ring that encircles the junction of the ARC and median eminence, which facilitates exposure of ARC neurons to the circulation. Most of the enmeshed neurons are both γ-aminobutyric acid-ergic and leptin-receptor positive, including a majority of Agouti-related-peptide neurons. Postnatal formation of the PNN-like structures coincides precisely with closure of the critical period for maturation of Agouti-related-peptide neurons and is dependent on input from circulating leptin, because postnatal ob/ob mice have reduced ARC PNN-like material that is restored by leptin administration during the critical period. We conclude that neurons crucial to metabolic homoeostasis are enmeshed in PNN-like structures and organized into a densely packed cluster situated circumferentially at the ARC–median eminence junction, where metabolically relevant humoral signals are sensed.

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Fig. 1: WFA labelling in the ventromedial ARC forms a ‘collar’ around the median eminence.
Fig. 2: PNNs enmesh GABAergic, LepRb+, AgRP/NPY neurons in the ARC.
Fig. 3: PNN formation in the ARC occurs during the lactation and periweaning period, corresponding to the maturation of AgRP neurons.
Fig. 4: Leptin-deficient ob/ob mice have impaired PNN formation during postnatal development that can be rescued by leptin administration during the critical period.

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. Additional detailed information on experimental design and reagents is available in the Reporting Summary.


  1. 1.

    Bouret, S. G., Draper, S. J. & Simerly, R. B. Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 304, 108–110 (2004).

    CAS  Article  Google Scholar 

  2. 2.

    Schwartz, M. W. et al. Obesity pathogenesis: an Endocrine Society scientific statement. Endocr. Rev. 38, 267–296 (2017).

    Article  Google Scholar 

  3. 3.

    Deem, J. D., Muta, K., Scarlett, J. M., Morton, G. J. & Schwartz, M. W. How should we think about the role of the brain in glucose homeostasis and diabetes? Diabetes 66, 1758–1765 (2017).

    CAS  Article  Google Scholar 

  4. 4.

    Kamitakahara, A., Bouyer, K., Wang, C. H. & Simerly, R. A critical period for the trophic actions of leptin on AgRP neurons in the arcuate nucleus of the hypothalamus. J. Comp. Neurol. 526, 133–145 (2018).

    CAS  Article  Google Scholar 

  5. 5.

    Pizzorusso, T. et al. Reactivation of ocular dominance plasticity in the adult visual cortex. Science 298, 1248–1251 (2002).

    CAS  Article  Google Scholar 

  6. 6.

    Hensch, T. K. Critical period plasticity in local cortical circuits. Nat. Rev. Neurosci. 6, 877–888 (2005).

    CAS  Article  Google Scholar 

  7. 7.

    Wiesel, T. N. & Hubel, D. H. Single-cell responses in striate cortex of kittens deprived of vision in one eye. J. Neurophysiol. 26, 1003–1017 (1963).

    CAS  Article  Google Scholar 

  8. 8.

    Carulli, D. et al. Animals lacking link protein have attenuated perineuronal nets and persistent plasticity. Brain 133, 2331–2347 (2010).

    Article  Google Scholar 

  9. 9.

    Kwok, J. C., Dick, G., Wang, D. & Fawcett, J. W. Extracellular matrix and perineuronal nets in CNS repair. Dev. Neurobiol. 71, 1073–1089 (2011).

    CAS  Article  Google Scholar 

  10. 10.

    Balmer, T. S., Carels, V. M., Frisch, J. L. & Nick, T. A. Modulation of perineuronal nets and parvalbumin with developmental song learning. J. Neurosci. 29, 12878–12885 (2009).

    CAS  Article  Google Scholar 

  11. 11.

    Gogolla, N., Caroni, P., Lüthi, A. & Herry, C. Perineuronal nets protect fear memories from erasure. Science 325, 1258–1261 (2009).

    CAS  Article  Google Scholar 

  12. 12.

    Nowicka, D., Soulsby, S., Skangiel-Kramska, J. & Glazewski, S. Parvalbumin-containing neurons, perineuronal nets and experience-dependent plasticity in murine barrel cortex. Eur. J. Neurosci. 30, 2053–2063 (2009).

    Article  Google Scholar 

  13. 13.

    Beurdeley, M. et al. Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex. J. Neurosci. 32, 9429–9437 (2012).

    CAS  Article  Google Scholar 

  14. 14.

    Miyata, S., Komatsu, Y., Yoshimura, Y., Taya, C. & Kitagawa, H. Persistent cortical plasticity by upregulation of chondroitin 6-sulfation. Nat. Neurosci. 15, 414–422 (2012).

    CAS  Article  Google Scholar 

  15. 15.

    Maeda, N. Structural variation of chondroitin sulfate and its roles in the central nervous system. Cent. Nerv. Syst. Agents Med. Chem. 10, 22–31 (2010).

    CAS  Article  Google Scholar 

  16. 16.

    Ahima, R. S., Prabakaran, D. & Flier, J. S. Postnatal leptin surge and regulation of circadian rhythm of leptin by feeding: implications for energy homeostasis and neuroendocrine function. J. Clin. Invest. 101, 1020–1027 (1998).

  17. 17.

    Baquero, A. F. et al. Developmental switch of leptin signaling in arcuate nucleus neurons. J. Neurosci. 34, 9982–9994 (2014).

    Article  Google Scholar 

  18. 18.

    Bouret, S. G. et al. Hypothalamic neural projections are permanently disrupted in diet-induced obese rats. Cell Metab. 7, 179–185 (2008).

    CAS  Article  Google Scholar 

  19. 19.

    Glavas, M. M. et al. Early overnutrition results in early-onset arcuate leptin resistance and increased sensitivity to high-fat diet. Endocrinology 151, 1598–1610 (2010).

    CAS  Article  Google Scholar 

  20. 20.

    Vogt, M. C. et al. Neonatal insulin action impairs hypothalamic neurocircuit formation in response to maternal high-fat feeding. Cell 156, 495–509 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    Bayol, S. A., Simbi, B. H. & Stickland, N. C. A maternal cafeteria diet during gestation and lactation promotes adiposity and impairs skeletal muscle development and metabolism in rat offspring at weaning. J. Physiol. 567, 951–961 (2005).

    CAS  Article  Google Scholar 

  22. 22.

    Gorski, J. N., Dunn-Meynell, A. A., Hartman, T. G. & Levin, B. E. Postnatal environment overrides genetic and prenatal factors influencing offspring obesity and insulin resistance. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R768–R778 (2006).

    CAS  Article  Google Scholar 

  23. 23.

    Carstens, K. E., Phillips, M. L., Pozzo-Miller, L., Weinberg, R. J. & Dudek, S. M. Perineuronal nets suppress plasticity of excitatory synapses on CA2 pyramidal neurons. J. Neurosci. 36, 6312–6320 (2016).

    CAS  Article  Google Scholar 

  24. 24.

    Köppe, G., Brückner, G., Härtig, W., Delpech, B. & Bigl, V. Characterization of proteoglycan-containing perineuronal nets by enzymatic treatments of rat brain sections. Histochem. J. 29, 11–20 (1997).

    Article  Google Scholar 

  25. 25.

    Bäckberg, M., Collin, M., Ovesjö, M. L. & Meister, B. Chemical coding of GABA(B) receptor-immunoreactive neurones in hypothalamic regions regulating body weight. J. Neuroendocrinol. 15, 1–14 (2003).

    Article  Google Scholar 

  26. 26.

    Morrison, C. D., Morton, G. J., Niswender, K. D., Gelling, R. W. & Schwartz, M. W. Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling. Am. J. Physiol. Endocrinol. Metab. 289, E1051–E1057 (2005).

    CAS  Article  Google Scholar 

  27. 27.

    McRae, P. A., Rocco, M. M., Kelly, G., Brumberg, J. C. & Matthews, R. T. Sensory deprivation alters aggrecan and perineuronal net expression in the mouse barrel cortex. J. Neurosci. 27, 5405–5413 (2007).

    CAS  Article  Google Scholar 

  28. 28.

    Nakamura, M. et al. Expression of chondroitin sulfate proteoglycans in barrel field of mouse and rat somatosensory cortex. Brain Res. 1252, 117–129 (2009).

    CAS  Article  Google Scholar 

  29. 29.

    Dityatev, A. et al. Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets. Dev. Neurobiol. 67, 570–588 (2007).

    CAS  Article  Google Scholar 

  30. 30.

    Luquet, S., Perez, F. A., Hnasko, T. S. & Palmiter, R. D. NPY/AgRP neurons are essential for feeding in adult mice but can be ablated in neonates. Science 310, 683–685 (2005).

    CAS  Article  Google Scholar 

  31. 31.

    Xu, J. et al. Genetic identification of leptin neural circuits in energy and glucose homeostases. Nature 556, 505–509 (2018).

    CAS  Article  Google Scholar 

  32. 32.

    de Winter, F. et al. The chemorepulsive protein semaphorin 3A and perineuronal net-mediated plasticity. Neural Plast. 2016, 3679545 (2016).

    CAS  Article  Google Scholar 

  33. 33.

    Barker, D. J. The fetal and infant origins of adult disease. BMJ 301, 1111 (1990).

    CAS  Article  Google Scholar 

  34. 34.

    Ravelli, G. P., Stein, Z. A. & Susser, M. W. Obesity in young men after famine exposure in utero and early infancy. N. Engl. J. Med. 295, 349–353 (1976).

    CAS  Article  Google Scholar 

  35. 35.

    Ravelli, A. C. et al. Glucose tolerance in adults after prenatal exposure to famine. Lancet 351, 173–177 (1998).

    CAS  Article  Google Scholar 

  36. 36.

    Vickers, M. H. et al. Neonatal leptin treatment reverses developmental programming. Endocrinology 146, 4211–4216 (2005).

    CAS  Article  Google Scholar 

  37. 37.

    Miyata, S. & Kitagawa, H. Formation and remodeling of the brain extracellular matrix in neural plasticity: roles of chondroitin sulfate and hyaluronan. Biochim. Biophys. Acta Gen. Subj. 1861, 2420–2434 (2017).

    CAS  Article  Google Scholar 

  38. 38.

    Saghatelyan, A. K. et al. Reduced perisomatic inhibition, increased excitatory transmission, and impaired long-term potentiation in mice deficient for the extracellular matrix glycoprotein tenascin-R. Mol. Cell. Neurosci. 17, 226–240 (2001).

    CAS  Article  Google Scholar 

  39. 39.

    Hylin, M. J., Orsi, S. A., Moore, A. N. & Dash, P. K. Disruption of the perineuronal net in the hippocampus or medial prefrontal cortex impairs fear conditioning. Learn. Mem. 20, 267–273 (2013).

    CAS  Article  Google Scholar 

  40. 40.

    Thaler, J. P. et al. Obesity is associated with hypothalamic injury in rodents and humans. J. Clin. Invest. 122, 153–162 (2012).

    CAS  Article  Google Scholar 

  41. 41.

    Douglass, J. D., Dorfman, M. D., Fasnacht, R., Shaffer, L. D. & Thaler, J. P. Astrocyte IKKβ/NF-κB signaling is required for diet-induced obesity and hypothalamic inflammation. Mol. Metab. 6, 366–373 (2017).

    CAS  Article  Google Scholar 

  42. 42.

    Valdearcos, M. et al. Microglial inflammatory signaling orchestrates the hypothalamic immune response to dietary excess and mediates obesity susceptibility. Cell Metab. 26, 185–197.e3 (2017).

    CAS  Article  Google Scholar 

  43. 43.

    Balland, E. et al. Hypothalamic tanycytes are an ERK-gated conduit for leptin into the brain. Cell Metab. 19, 293–301 (2014).

    CAS  Article  Google Scholar 

  44. 44.

    Gao, Y. et al. Hormones and diet, but not body weight, control hypothalamic microglial activity. Glia 62, 17–25 (2014).

    Article  Google Scholar 

  45. 45.

    Tamamaki, N. et al. Green fluorescent protein expression and colocalization with calretinin, parvalbumin, and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60–79 (2003).

    CAS  Article  Google Scholar 

  46. 46.

    Leshan, R. L., Björnholm, M., Münzberg, H. & Myers, M. G.Jr. Leptin receptor signaling and action in the central nervous system. Obesity (Silver Spring) 14, 208S–212S (2006).

    CAS  Article  Google Scholar 

  47. 47.

    Madisen, L. et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

    CAS  Article  Google Scholar 

  48. 48.

    van den Pol, A. N. et al. Neuromedin B and gastrin-releasing peptide excite arcuate nucleus neuropeptide Y neurons in a novel transgenic mouse expressing strong renilla green fluorescent protein in NPY neurons. J. Neurosci. 29, 4622–4639 (2009).

    CAS  Article  Google Scholar 

  49. 49.

    Cowley, M. A. et al. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411, 480–484 (2001).

    CAS  Article  Google Scholar 

  50. 50.

    Mirzadeh, Z., Doetsch, F., Sawamoto, K., Wichterle, H. & Alvarez-Buylla, A. The subventricular zone en-face: wholemount staining and ependymal flow. J. Vis. Exp. 39, 1938 (2010).

    Google Scholar 

  51. 51.

    Doetsch, F., García-Verdugo, J. M. & Alvarez-Buylla, A. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17, 5046–5061 (1997).

    CAS  Article  Google Scholar 

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The authors are grateful to the original providers of the transgenic mouse lines used in this work: N. Tamamaki (GAD67-GFP), M. Myers (LepRb-Cre), H. Zeng (Ai14), B. Lowell (NPY-GFP) and M. Low (POMC-GFP). This work was supported by the US National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (grant nos. DK108596 (Z.M.), DK114474 (J.M.S.), DK083042 (M.W.S.), DK090320 (M.W.S.) and DK101997 (M.W.S.)), the National Institute of Neurological Disorders and Stroke Neurosurgeon Research Career Development Program K Award (Z.M.), the American Diabetes Association (grant no. 7–11-BS-179 (L.Z.)), the Russell Berrie Foundation (R.H.) and the Barrow Neurological Foundation (grant no. 18-0025-30-05 (Z.M.)).

Author information




Z.M., K.M.A. and M.W.S. conceived and designed the study; Z.M., K.M.A., E.C., V.H.P., J.M.S., J.M.B., R.H., M.E.M., H.T.N., J.M.G.V., L.M.Z. and M.W.S. acquired, analysed and interpreted the data; Z.M. and M.W.S. drafted and revised the manuscript. All authors approved the final version of the manuscript.

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Correspondence to Zaman Mirzadeh or Michael W. Schwartz.

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Supplementary information

Supplementary Information

Supplementary Figures. 1–9

Reporting Summary

Supplementary Video 1

Imaris three-dimensional (3D) surface rendering of an isolated PNN-enmeshed (red) GAD67-GFP+ ARC neuron (green). This rendering was performed on a 63× image taken from a coronal section, through the ARC of a GAD67-GFP mouse, stained with WFA (red) and GFP antibody (green). This 3D image is representative of renderings performed on five other GAD67-GFP+ ARC neurons

Supplementary Video 2

Imaris 3D surface rendering of an isolated PNN-enmeshed (red) GAD67-GFP+ V1 interneuron (green). This rendering was performed on a 63× image taken from a coronal section, through V1 of a GAD67-GFP mouse, stained with WFA (red) and GFP antibody (green). This 3D image is representative of renderings performed on three other GAD67-GFP+ V1 neurons.

Supplementary Video 3

Imaris 3D surface rendering of an isolated PNN-enmeshed (red) Npy-GFP+ ARC neuron (green). This rendering was performed on a 63× image taken from a coronal section, through the ARC of a Npy-GFP mouse, stained with WFA (red) and GFP antibody (green). This 3D image is representative of renderings performed on five other NPY-GFP+ neurons.

Supplementary Video 4

Imaris 3D surface rendering of an isolated PNN-enmeshed (red) NPY+ human ARC neuron (green). This rendering was performed on a 63× image taken from a coronal section, through the ARC of a human brain, stained with WFA (red) and NPY antibody (green). This 3D image is representative of renderings performed on four other NPY+ human ARC neurons.

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Mirzadeh, Z., Alonge, K.M., Cabrales, E. et al. Perineuronal net formation during the critical period for neuronal maturation in the hypothalamic arcuate nucleus. Nat Metab 1, 212–221 (2019).

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