Brain Structure and Function

, Volume 223, Issue 3, pp 1133–1148 | Cite as

Perineuronal nets labeled by monoclonal antibody VC1.1 ensheath interneurons expressing parvalbumin and calbindin in the rat amygdala

  • Alexander J. McDonald
  • Patricia G. Hamilton
  • Colin J. Barnstable
Original Article


Perineuronal nets (PNNs) are specialized condensations of extracellular matrix that ensheath particular neuronal subpopulations in the brain and spinal cord. PNNs regulate synaptic plasticity, including the encoding of fear memories by the amygdala. The present immunohistochemical investigation studied PNN structure and distribution, as well as the neurochemistry of their ensheathed neurons, in the rat amygdala using monoclonal antibody VC1.1, which recognizes a glucuronic acid 3-sulfate glycan associated with PNNs in the cerebral cortex. VC1.1+ PNNs surrounded the cell bodies and dendrites of a subset of nonpyramidal neurons in cortex-like portions of the amygdala (basolateral amygdalar complex, cortical nuclei, nucleus of the lateral olfactory tract, and amygdalohippocampal region). There was also significant neuropilar VC1.1 immunoreactivity, whose density varied in different amygdalar nuclei. Cell counts in the basolateral nucleus revealed that virtually all neurons ensheathed by VC1.1+ PNNs were parvalbumin-positive (PV+) interneurons, and these VC1.1+/PV+ cells constituted 60% of all PV+ interneurons, including all of the larger PV+ neurons. Approximately 70% of VC1.1+ neurons were calbindin-positive (CB+), and these VC1.1+/CB+ cells constituted about 40% of all CB+ neurons. Colocalization of VC1.1 with Vicia villosa agglutinin (VVA) binding, which stains terminal N-acetylgalactosamines, revealed that VC1.1+ PNNs were largely a subset of VVA+ PNNs. This investigation provides baseline data regarding PNNs in the rat which should be useful for future studies of their function in this species.


Extracellular matrix Perineuronal nets Amygdala Calcium-binding proteins Immunohistochemistry 



The authors thank Dr. Kenneth G. Baimbridge (University of British Columbia) for his generous donation of the PV and CB polyclonal antisera. This work was supported by NIH Grants R01MH104638 and R01NS19733.

Compliance with ethical standards

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Human and animal rights statement

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abo T, Balch CM (1981) A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J Immunol 127:1024–1029PubMedGoogle Scholar
  2. Alpár A, Gärtner U, Härtig W, Brückner G (2006) Distribution of pyramidal cells associated with perineuronal nets in the neocortex of rat. Brain Res 1120:13–22CrossRefPubMedGoogle Scholar
  3. Arimatsu Y, Naegele JR, Barnstable CJ (1987) Molecular markers of neuronal subpopulations in layers 4, 5, and 6 of cat primary visual cortex. J Neurosci 7:1250–1263PubMedGoogle Scholar
  4. Baker KD, Gray AR, Richardson R (2017) The development of perineuronal nets around parvalbumin gabaergic neurons in the medial prefrontal cortex and basolateral amygdala of rats. Behav Neurosci 131:289–303CrossRefPubMedGoogle Scholar
  5. Balmer TS (2016) Perineuronal nets enhance the excitability of fast-spiking neurons. eNeuro 3:1–13. doi: 10.1523/ENEURO.0112-16.2016 CrossRefGoogle Scholar
  6. Barnstable CJ, Kosaka T, Naegele JR, Arimatsu Y (1992) Molecular properties of GABAergic local-circuit neurons in the mammalian visual cortex. Prog Brain Res 90:503–522CrossRefPubMedGoogle Scholar
  7. Bekku Y, Rauch U, Ninomiya Y, Oohashi T (2009) Brevican distinctively assembles extracellular components at the large diameter nodes of Ranvier in the CNS. J Neurochem 108:1266–1276CrossRefPubMedGoogle Scholar
  8. Berretta S (2012) Extracellular matrix abnormalities in schizophrenia. Neuropharmacology 62:1584–1597CrossRefPubMedGoogle Scholar
  9. Berretta S, Pantazopoulos H, Markota M, Brown C, Batzianouli ET (2015) Losing the sugar coating: potential impact of perineuronal net abnormalities on interneurons in schizophrenia. Schizophr Res 167:18–27CrossRefPubMedPubMedCentralGoogle Scholar
  10. Bienvenu TC, Busti D, Magill PJ, Ferraguti F, Capogna M (2012) Cell-type-specific recruitment of amygdala interneurons to hippocampal theta rhythm and noxious stimuli in vivo. Neuron 74:1059–1074CrossRefPubMedGoogle Scholar
  11. Blosa M, Sonntag M, Jäger C, Weigel S, Seeger J, Frischknecht R, Seidenbecher CI, Matthews RT, Arendt T, Rübsamen R, Morawski M (2015) The extracellular matrix molecule brevican is an integral component of the machinery mediating fast synaptic transmission at the calyx of Held. J Physiol 593:4341–4360CrossRefPubMedPubMedCentralGoogle Scholar
  12. Brückner G, Brauer K, Härtig W, Wolff JR, Rickmann MJ, Derouiche A, Delpech B, Girard N, Oertel WH, Reichenbach A (1993) Perineuronal nets provide a polyanionic, glia-associated form of microenvironment around certain neurons in many parts of the rat brain. Glia 8:183–200CrossRefPubMedGoogle Scholar
  13. Brückner G, Grosche J, Schmidt S, Härtig W, Margolis RU, Delpech B, Seidenbecher CI, Czaniera R, Schachner M (1999) Postnatal development of perineuronal nets in wild-type mice and in a mutant deficient in tenascin-R. J Comp Neurol 428:616–629CrossRefGoogle Scholar
  14. Bukalo O, Schachner M, Dityatev A (2001) Modification of extracellular matrix by enzymatic removal of chondroitin sulfate and by lack of tenascin-R differentially affects several forms of synaptic plasticity in the hippocampus. Neuroscience 104:359–369CrossRefPubMedGoogle Scholar
  15. Carulli D, Rhodes KE, Brown DJ, Bonnert TP, Pollack SJ, Oliver K, Strata P, Fawcett JW (2006) Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components. J Comp Neurol 494:559–577CrossRefPubMedGoogle Scholar
  16. Celio MR, Blümcke I (1994) Perineuronal nets—a specialized form of extracellular matrix in the adult nervous system. Brain Res Brain Res Rev 19:128–145CrossRefPubMedGoogle Scholar
  17. Celio MR, Spreafico R, De Biasi S, Vitellaro-Zuccarello L (1998) Perineuronal nets: past and present. Trends Neurosci 21:510–515CrossRefPubMedGoogle Scholar
  18. Condé F, Lund JS, Jacobowitz DM, Baimbridge KG, Lewis DA (1994) Local circuit neurons immunoreactive for calretinin, calbindin D-28k or parvalbumin in monkey prefrontal cortex: distribution and morphology. J Comp Neurol 341:95–116CrossRefPubMedGoogle Scholar
  19. Dauth S, Grevesse T, Pantazopoulos H, Campbell PH, Maoz BM, Berretta S, Parker KK (2016) Extracellular matrix protein expression is brain region dependent. J Comp Neurol 524:1309–1336CrossRefPubMedGoogle Scholar
  20. Dityatev A, Schachner M (2003) Extracellular matrix molecules and synaptic plasticity. Nat Rev Neurosci 4:456–468CrossRefPubMedGoogle Scholar
  21. Galtrey CM, Fawcett JW (2007) The role of chondroitin sulfate proteoglycans in regeneration and plasticity in the central nervous system. Brain Res Rev 54:1–18CrossRefPubMedGoogle Scholar
  22. Gogolla N, Caroni P, Lüthi A, Herry C (2009) Perineuronal nets protect fear memories from erasure. Science 325:1258–1261CrossRefPubMedGoogle Scholar
  23. Golgi C (1893) Intorno all’origine del quarto nervo cerebrale e una questione isto-fisiologica che a questo argomento si collega. Rendiconti della Reale Accademia dei Lincei (21 maggio) 2:443–450Google Scholar
  24. Hancock MB (1986) Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements. Am J Anat 175:343–352CrossRefPubMedGoogle Scholar
  25. Härtig W, Brückner G, Brauer K, Schmidt C, Bigl V (1995) Allocation of perineuronal nets and parvalbumin-, calbindin-D28k- and glutamic acid decarboxylase-immunoreactivity in the amygdala of the rhesus monkey. Brain Res 698:265–269CrossRefPubMedGoogle Scholar
  26. Härtig W, Derouiche A, Welt K, Brauer K, Grosche J, Mäder M, Reichenbach A, Brückner G (1999) Cortical neurons immunoreactive for the potassium channel Kv3.1b subunit are predominantly surrounded by perineuronal nets presumed as a buffering system for cations. Brain Res 842:15–29CrossRefPubMedGoogle Scholar
  27. Hensch TK (2005) Critical period plasticity in local cortical circuits. Nat Rev Neurosci 6:877–888CrossRefPubMedGoogle Scholar
  28. Holt DJ, Lebron-Milad K, Milad MR, Rauch SL, Pitman RK, Orr SP, Cassidy BS, Walsh JP, Goff DC (2009) Extinction memory is impaired in schizophrenia. Biol Psychiatry 65:455–463CrossRefPubMedGoogle Scholar
  29. Horii-Hayashi N, Sasagawa T, Matsunaga W, Nishi M (2015) Development and structural variety of the chondroitin sulfate proteoglycans-contained extracellular matrix in the mouse brain. Neural Plast 2015:256389CrossRefPubMedPubMedCentralGoogle Scholar
  30. Kemppainen S, Pitkänen A (2000) Distribution of parvalbumin, calretinin, and calbindin-D(28k) immunoreactivity in the rat amygdaloid complex and colocalization with gamma-aminobutyric acid. J Comp Neurol 426:441–467CrossRefPubMedGoogle Scholar
  31. Kizuka Y, Oka S (2012) Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate. Cell Mol Life Sci 69:4135–4147CrossRefPubMedGoogle Scholar
  32. Kosaka T, Heizmann CW, Barnstable CJ (1989) Monoclonal antibody VC1.1 selectively stains a population of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat cerebral cortex. Exp Brain Res 78:43–50CrossRefPubMedGoogle Scholar
  33. Kosaka T, Isogai K, Barnstable CJ, Heizmann CW (1990) Monoclonal antibody HNK-1 selectively stains a subpopulation of GABAergic neurons containing the calcium-binding protein parvalbumin in the rat cerebral cortex. Exp Brain Res 82:566–574CrossRefPubMedGoogle Scholar
  34. Lucas EK, Jegarl AM, Morishita H, Clem RL (2016) Multimodal and site-specific plasticity of amygdala parvalbumin interneurons after fear learning. Neuron 91:629–643CrossRefPubMedPubMedCentralGoogle Scholar
  35. Maeda N (2015) Proteoglycans and neuronal migration in the cerebral cortex during development and disease. Front Neurosci 9:98CrossRefPubMedPubMedCentralGoogle Scholar
  36. Mascagni F, McDonald AJ (2003) Immunohistochemical characterization of cholecystokinin containing neurons in the rat basolateral amygdala. Brain Res 976:171–184CrossRefPubMedGoogle Scholar
  37. Matthews RT, Kelly GM, Zerillo CA, Gray G, Tiemeyer M, Hockfield S (2002) Aggrecan glycoforms contribute to the molecular heterogeneity of perineuronal nets. J Neurosci 22:7536–7547PubMedGoogle Scholar
  38. McDonald AJ (1982) Neurons of the lateral and basolateral amygdaloid nuclei: a Golgi study in the rat. J Comp Neurol 212:293–312CrossRefPubMedGoogle Scholar
  39. McDonald AJ (1992) Cell types and intrinsic connections of the amygdala. In: Aggleton JP (ed) the amygdala. Wiley-Liss, New York, pp 67–96Google Scholar
  40. McDonald AJ (1997) Calbindin-D28k immunoreactivity in the rat amygdala. J Comp Neurol 383:231–244CrossRefPubMedGoogle Scholar
  41. McDonald AJ, Betette RL (2001) Parvalbumin-containing neurons in the rat basolateral amygdala: morphology and co-localization of calbindin-D(28k). Neuroscience 102:413–425CrossRefPubMedGoogle Scholar
  42. McDonald AJ, Culberson JL (1981) Neurons of the basolateral amygdala: a Golgi study in the opossum (Didelphis virginiana). Am J Anat 162:327–342CrossRefPubMedGoogle Scholar
  43. McDonald AJ, Mascagni F (2001) Colocalization of calcium-binding proteins and GABA in neurons of the rat basolateral amygdala. Neuroscience 105:681–693CrossRefPubMedGoogle Scholar
  44. McDonald AJ, Mascagni F (2002) Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala. Brain Res 943:237–244CrossRefPubMedGoogle Scholar
  45. McDonald AJ, Mascagni F (2006) Differential expression of Kv3.1b and Kv3.2 potassium channel subunits in interneurons of the basolateral amygdala. Neuroscience 138:537–547CrossRefPubMedGoogle Scholar
  46. McDonald AJ, Mascagni F, Mania I, Rainnie DG (2005) Evidence for a perisomatic innervation of parvalbumin-containing interneurons by individual pyramidal cells in the basolateral amygdala. Brain Res 1035:32–40CrossRefPubMedGoogle Scholar
  47. Morikawa S, Ikegaya Y, Narita M, Tamura H (2017) Activation of perineuronal net-expressing excitatory neurons during associative memory encoding and retrieval. Sci Rep 7:46024CrossRefPubMedPubMedCentralGoogle Scholar
  48. Mueller AL, Davis A, Sovich S, Carlson SS, Robinson FR (2016) Distribution of n-acetylgalactosamine-positive perineuronal nets in the macaque brain: anatomy and Implications. Neural Plast 2016:6021428CrossRefPubMedPubMedCentralGoogle Scholar
  49. Naegele JR, Barnstable CJ (1991) A carbohydrate epitope defined by monoclonal antibody VC1.1 is found on N-CAM and other cell adhesion molecules. Brain Res 559:118–129CrossRefPubMedGoogle Scholar
  50. Naegele JR, Katz LC (1990) Cell surface molecules containing N-acetylgalactosamine are associated with basket cells and neurogliaform cells in cat visual cortex. J Neurosci 10:540–557PubMedGoogle Scholar
  51. Naegele JR, Arimatsu Y, Schwartz P, Barnstable CJ (1988) Selective staining of a subset of GABAergic neurons in cat visual cortex by monoclonal antibody VC1.1. J Neurosci 8:79–89PubMedGoogle Scholar
  52. Pantazopoulos H, Berretta S (2016) In sickness and in health: perineuronal nets and synaptic plasticity in psychiatric disorders. Neural Plast 2016:9847696CrossRefPubMedGoogle Scholar
  53. Pantazopoulos H, Lange N, Hassinger L, Berretta S (2006) Subpopulations of neurons expressing parvalbumin in the human amygdala. J Comp Neurol 496:706–722CrossRefPubMedPubMedCentralGoogle Scholar
  54. Pantazopoulos H, Murray EA, Berretta S (2008) Total number, distribution, and phenotype of cells expressing chondroitin sulfate proteoglycans in the normal human amygdala. Brain Res 1207:84–95CrossRefPubMedPubMedCentralGoogle Scholar
  55. Pantazopoulos H, Woo TU, Lim MP, Lange N, Berretta S (2010) Extracellular matrix-glial abnormalities in the amygdala and entorhinal cortex of subjects diagnosed with schizophrenia. Arch Gen Psychiatry 67:155–166CrossRefPubMedPubMedCentralGoogle Scholar
  56. Pantazopoulos H, Markota M, Jaquet F, Ghosh D, Wallin A, Santos A, Caterson B, Berretta S (2015) Aggrecan and chondroitin-6-sulfate abnormalities in schizophrenia and bipolar disorder: a postmortem study on the amygdala. Transl Psychiatry 5:e496CrossRefPubMedPubMedCentralGoogle Scholar
  57. Pape HC, Narayanan RT, Smid J, Stork O, Seidenbecher T (2005) Theta activity in neurons and networks of the amygdala related to long-term fear memory. Hippocampus 15:874–880CrossRefPubMedGoogle Scholar
  58. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates. Academic Press, New YorkGoogle Scholar
  59. Rainnie DG, Mania I, Mascagni F, McDonald AJ (2006) Physiological and morphological characterization of parvalbumin-containing interneurons of the rat basolateral amygdala. J Comp Neurol 498:142–161CrossRefPubMedGoogle Scholar
  60. Ren JQ, Heizmann CW, Kosaka T (1994) Regional difference in the distribution of parvalbumin-containing neurons immunoreactive for monoclonal antibody HNK-1 in the mouse cerebral cortex. Neurosci Lett 166:221–225CrossRefPubMedGoogle Scholar
  61. Saghatelyan AK, Gorissen S, Albert M, Hertlein B, Schachner M, Dityatev A (2000) The extracellular matrix molecule tenascin-R and its HNK-1 carbohydrate modulate perisomatic inhibition and long-term potentiation in the CA1 region of the hippocampus. Eur J Neurosci 12:3331–33342CrossRefPubMedGoogle Scholar
  62. Saghatelyan AK, Dityatev A, Schmidt S, Schuster T, Bartsch U, Schachner M (2001) Reduced perisomatic inhibition, increased excitatory transmission, and impaired long-term potentiation in mice deficient for the extracellular matrix glycoprotein tenascin-R. Mol Cell Neurosci 17:226–240CrossRefPubMedGoogle Scholar
  63. Smith PD, Coulson-Thomas VJ, Foscarin S, Kwok JC, Fawcett JW (2015) “GAG-ing with the neuron”: the role of glycosaminoglycan patterning in the central nervous system. Exp Neurol 274(Pt B):100–114CrossRefPubMedGoogle Scholar
  64. Sorg BA, Berretta S, Blacktop JM, Fawcett JW, Kitagawa H, Kwok JC, Miquel M (2016) Casting a wide net: role of perineuronal nets in neural plasticity. J Neurosci 36:11459–11468CrossRefPubMedPubMedCentralGoogle Scholar
  65. Trouche S, Sasaki JM, Tu T, Reijmers LG (2013) Fear extinction causes target-specific remodeling of perisomatic inhibitory synapses. Neuron 80:1054–1065CrossRefPubMedGoogle Scholar
  66. Tsien RY (2013) Very long-term memories may be stored in the pattern of holes in the perineuronal net. Proc Natl Acad Sci USA 110:12456–12461CrossRefPubMedPubMedCentralGoogle Scholar
  67. Umemori J, Winkel F, Castrén E, Karpova NN (2015) Distinct effects of perinatal exposure to fluoxetine or methylmercury on parvalbumin and perineuronal nets, the markers of critical periods in brain development. Int J Dev Neurosci 44:55–64CrossRefPubMedGoogle Scholar
  68. Vereczki VK, Veres JM, Müller K, Nagy GA, Rácz B, Barsy B, Hájos N (2016) Synaptic organization of perisomatic GABAergic inputs onto the principal cells of the mouse basolateral amygdala. Front Neuroanat 10:20CrossRefPubMedPubMedCentralGoogle Scholar
  69. Veres JM, Nagy GA, Hájos N (2017) Perisomatic GABAergic synapses of basket cells effectively control principal neuron activity in amygdala networks. Elife 6:e20721CrossRefPubMedPubMedCentralGoogle Scholar
  70. Viapiano MS, Matthews RT (2006) From barriers to bridges: chondroitin sulfate proteoglycans in neuropathology. Trends Mol Med 12:488–496CrossRefPubMedGoogle Scholar
  71. Wang D, Fawcett J (2012) The perineuronal net and the control of CNS plasticity. Cell Tissue Res 349:147–160CrossRefPubMedGoogle Scholar
  72. Weber P, Bartsch U, Rasband MN, Czaniera R, Lang Y, Bluethmann H, Margolis RU, Levinson SR, Shrager P, Montag D, Schachner M (1999) Mice deficient for tenascin-R display alterations of the extracellular matrix and decreased axonal conduction velocities in the CNS. J Neurosci 19:4245–4262PubMedGoogle Scholar
  73. Wegner F, Härtig W, Bringmann A, Grosche J, Wohlfarth K, Zuschratter W, Brückner G (2003) Diffuse perineuronal nets and modified pyramidal cells immunoreactive for glutamate and the GABA(A) receptor alpha1 subunit form a unique entity in rat cerebral cortex. Exp Neurol 184:705–714CrossRefPubMedGoogle Scholar
  74. Wolff SB, Gründemann J, Tovote P, Krabbe S, Jacobson GA, Müller C, Herry C, Ehrlich I, Friedrich RW, Letzkus JJ, Lüthi A (2014) Amygdala interneuron subtypes control fear learning through disinhibition. Nature 509:453–458CrossRefPubMedGoogle Scholar
  75. Woodruff AR, Sah P (2007) Networks of parvalbumin-positive interneurons in the basolateral amygdala. J Neurosci 27:553–563CrossRefPubMedGoogle Scholar
  76. Woodson W, Farb CR, Ledoux JE (2000) Afferents from the auditory thalamus synapse on inhibitory interneurons in the lateral nucleus of the amygdala. Synapse 38:124–137CrossRefPubMedGoogle Scholar
  77. Xue YX, Xue LF, Liu JF, He J, Deng JH, Sun SC, Han HB, Luo YX, Xu LZ, Wu P, Lu L (2014) Depletion of perineuronal nets in the amygdala to enhance the erasure of drug memories. J Neurosci 34:6647–6658CrossRefPubMedGoogle Scholar
  78. Yamada J, Jinno S (2015) Subclass-specific formation of perineuronal nets around parvalbumin-expressing GABAergic neurons in Ammon’s horn of the mouse hippocampus. J Comp Neurol 523:790–804CrossRefPubMedGoogle Scholar
  79. Yamamoto M, Marshall P, Hemmendinger LM, Boyer AB, Caviness VS Jr (1988) Distribution of glucuronic acid-and-sulfate-containing glycoproteins in the central nervous system of the adult mouse. Neurosci Res 5:273–298CrossRefPubMedGoogle Scholar
  80. Zaremba S, Naegele JR, Barnstable CJ, Hockfield S (1990) Neuronal subsets express multiple high-molecular-weight cell-surface glycoconjugates defined by monoclonal antibodies Cat-301 and VC1.1. J Neurosci 10:2985–2995PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Alexander J. McDonald
    • 1
  • Patricia G. Hamilton
    • 1
  • Colin J. Barnstable
    • 2
  1. 1.Department of Pharmacology, Physiology and NeuroscienceUniversity of South Carolina School of MedicineColumbiaUSA
  2. 2.Department of Neural and Behavioral SciencesThe Pennsylvania State University College of MedicineHersheyUSA

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