Separate Gene Transfers into Pre- and Postsynaptic Neocortical Neurons Connected by mGluR5-Containing Synapses

  • Aarti Nagayach
  • Anshuman Singh
  • Alfred I. GellerEmail author


mGluR5-containing synapses have essential roles in synaptic plasticity, circuit physiology, and learning, and dysfunction at these synapses is implicated in specific neurological disorders. As mGluR5-containing synapses are embedded in large and complex distributed circuits containing many neuron and synapse types, it is challenging to elucidate the roles of these synapses and to develop treatments for the associated disorders. Thus, it would be advantageous to deliver different genes into pre- and postsynaptic neurons connected by a mGluR5-containing synapse. Here, we develop this capability: The first gene transfer, into the presynaptic neurons, uses standard techniques to deliver a vector that expresses a synthetic peptide neurotransmitter. This peptide neurotransmitter has three domains: a dense core vesicle sorting domain, a mGluR5-binding domain composed of a single-chain variable fragment anti-mGluR5, and the His tag. Upon release, this peptide neurotransmitter binds to mGluR5, predominately located on the postsynaptic neurons. Selective gene transfer into these neurons uses antibody-mediated, targeted gene transfer and anti-His tag antibodies, as the synthetic peptide neurotransmitter contains the His tag. For the model system, we studied the connection between neurons in two neocortical areas: postrhinal and perirhinal cortices. Targeted gene transfer was over 80% specific for mGluR5-containing synapses, but untargeted gene transfer was only ~ 15% specific for these synapses. This technology may enable studies on the roles of mGluR5-containing neurons and synapses in circuit physiology and learning and support gene therapy treatments for specific disorders that involve dysfunction at these synapses.


mGluR5 Synapse Synthetic peptide neurotransmitter Targeted gene transfer Herpes simplex virus vector Single-chain variable fragment 



Assistance from the Research to Prevent Blindness and the Lions Eye Foundation is gratefully acknowledged.

Author Contributions

A.N. is responsible for the experimental design, gene transfer surgery, perfusions, brain sectioning, immunofluorescent staining, microscopy, cell counts, and figure preparation; A.S. for the vector packaging, purification, and titering; and A.I.G. for the project design, supervision, data analysis, statistical analysis, figure preparation, manuscript writing, and editing.

Funding Information

This work was supported by NIH Grant NS086960 (AIG).

Compliance with Ethical Standards

All animal care and experimental procedures were approved by the Louisiana State University Health Science Center New Orleans IACUC.

Conflict of Interest

A.I.G. has equity in Alkermes Inc.

Supplementary material

12031_2019_1317_MOESM1_ESM.pdf (33 kb)
ESM 1 (PDF 33 kb)


  1. Agster KL, Burwell RD (2009) Cortical efferents of the perirhinal, postrhinal, and entorhinal cortices of the rat. Hippocampus. 19:1159–1186CrossRefGoogle Scholar
  2. Bashir ZI, Bortolotto ZA, Davies CH, Berretta N, Irving AJ, Seal AJ, Henley JM, Jane DE, Watkins JC, Collingridge GL (1993) Induction of LTP in the hippocampus needs synaptic activation of glutamate metabotropic receptors. Nature. 363:347–350CrossRefGoogle Scholar
  3. Bergman I, Whitaker-Dowling P, Gao Y, Griffin JA, Watkins SC (2003) Vesicular stomatitis virus expressing a chimeric Sindbis glycoprotein containing an Fc antibody binding domain targets to Her2/neu overexpressing breast cancer cells. Virology. 316:337–347CrossRefGoogle Scholar
  4. Burwell RD, Amaral DG (1998a) Cortical afferents of the perirhinal, postrhinal, and entorhinal cortices of the rat. J Comp Neurol 398:179–205CrossRefGoogle Scholar
  5. Burwell RD, Amaral DG (1998b) Perirhinal and postrhinal cortices of the rat: interconnectivity and connections with the entorhinal cortex. J Comp Neurol 391:293–321CrossRefGoogle Scholar
  6. Burwell RD (2000) The parahippocampal region: corticocortical connectivity. Ann NY Acad Sci 911:25–42CrossRefGoogle Scholar
  7. Cao H, Zhang GR, Geller AI (2010) Antibody-mediated targeted gene transfer to NMDA NR1-containing neurons in rat neocortex by helper virus-free HSV-1 vector particles containing a chimeric HSV-1 glycoprotein C–staphylococcus A protein. Brain Res 1351:1–12CrossRefGoogle Scholar
  8. Cao H, Zhang GR, Geller AI (2011) Antibody-mediated targeted gene transfer of helper virus-free HSV-1 vectors to rat neocortical neurons that contain either NMDA receptor 2A or 2B subunits. Brain Res 1415:127–135CrossRefGoogle Scholar
  9. Cool DR, Loh YP (1994) Identification of a sorting signal for the regulated secretory pathway at the N-terminus of pro-opiomelanocortin. Biochimie. 76:265–270CrossRefGoogle Scholar
  10. Cool DR, Fenger M, Snell CR, Loh YP (1995) Identification of the sorting signal motif within pro-opiomelanocortin for the regulated secretory pathway. J Biol Chem 270:8723–8729CrossRefGoogle Scholar
  11. Courel M, Vasquez MS, Hook VY, Mahata SK, Taupenot L (2008) Sorting of the neuroendocrine secretory protein secretogranin II into the regulated secretory pathway: role of N- and C-terminal alpha-helical domains. J Biol Chem 283:11807–11822CrossRefGoogle Scholar
  12. Dikeakos JD, Reudelhuber TL (2007) Sending proteins to dense core secretory granules: still a lot to sort out. J Cell Biol 177:191–196CrossRefGoogle Scholar
  13. Dudai Y (1989) The neurobiology of memory. Oxford Univ. Press, OxfordGoogle Scholar
  14. Dymecki SM, Kim JC (2007) Molecular neuroanatomy’s “three Gs”: a primer. Neuron. 54:17–34CrossRefGoogle Scholar
  15. Fatemi SH, Folsom TD (2015) GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism. Schizophr Res 167:42–56CrossRefGoogle Scholar
  16. Feng L, Kwon O, Lee B, Oh WC, Kim J (2014) Using mammalian GFP reconstitution across synaptic partners (mGRASP) to map synaptic connectivity in the mouse brain. Nat Protoc 9:2425–2437CrossRefGoogle Scholar
  17. Fenno L, Yizhar O, Deisseroth K (2011) The development and application of optogenetics. Annu Rev Neurosci 34:389–412CrossRefGoogle Scholar
  18. Fraefel C, Song S, Lim F, Lang P, Yu L, Wang Y, Wild P, Geller AI (1996) Helper virus-free transfer of herpes simplex virus type 1 plasmid vectors into neural cells. J Virol 70:7190–7197Google Scholar
  19. Gao Q, Sun M, Wang X, Geller AI (2007) Isolation of an enhancer from the rat tyrosine hydroxylase promoter that supports long-term, neuronal-specific expression from a neurofilament promoter, in a helper virus-free HSV-1 vector system. Brain Res 1130:1–16CrossRefGoogle Scholar
  20. Gerdes HH et al (1989) The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation. J Biol Chem 264:12009–12015Google Scholar
  21. Hermans E, Challiss RA (2001) Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors. Biochem J 359:465–484CrossRefGoogle Scholar
  22. Homayoun H, Moghaddam B (2010) Group 5 metabotropic glutamate receptors: role in modulating cortical activity and relevance to cognition. Eur J Pharmacol 639:33–39CrossRefGoogle Scholar
  23. Huston JS et al (1991) Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol 203:46–88CrossRefGoogle Scholar
  24. Kameda H, Furuta T, Matsuda W, Ohira K, Nakamura K, Hioki H, Kaneko T (2008) Targeting green fluorescent protein to dendritic membrane in central neurons. Neurosci Res 61:79–91CrossRefGoogle Scholar
  25. Kim J, Zhao T, Petralia RS, Yu Y, Peng H, Myers E, Magee JC (2011) mGRASP enables mapping mammalian synaptic connectivity with light microscopy. Nat Methods 9:96–102CrossRefGoogle Scholar
  26. Kozak M (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 44:283–292CrossRefGoogle Scholar
  27. Kumar A, Dhull DK, Mishra PS (2015) Therapeutic potential of mGluR5 targeting in Alzheimer’s disease. Front Neurosci 9:215Google Scholar
  28. Linden DJ (1994) Long-term synaptic depression in the mammalian brain. Neuron. 12:457–472CrossRefGoogle Scholar
  29. Lo L, Anderson DJ (2011) A cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron. 72:938–950CrossRefGoogle Scholar
  30. Lujan R et al (1996) Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus. Eur J Neurosci 8:1488–1500CrossRefGoogle Scholar
  31. Luo L, Callaway EM, Svoboda K (2008) Genetic dissection of neural circuits. Neuron. 57:634–660CrossRefGoogle Scholar
  32. Morizono K, Bristol G, Xie YM, Kung SKP, Chen ISY (2001) Antibody-directed targeting of retroviral vectors via cell surface antigens. J Virol 75:8016–8020CrossRefGoogle Scholar
  33. Morizono K, Chen IS (2005) Targeted gene delivery by intravenous injection of retroviral vectors. Cell Cycle 4:854–856CrossRefGoogle Scholar
  34. Morizono K, Xie Y, Ringpis GE, Johnson M, Nassanian H, Lee B, Wu L, Chen ISY (2005) Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection. Nat Med 11:346–352CrossRefGoogle Scholar
  35. Moskal JR, Yamamoto H, Colley PA (2001) The use of antibody engineering to create novel drugs that target N-methyl-D-aspartate receptors. Curr Drug Targets 2:331–345CrossRefGoogle Scholar
  36. Moskal JR, Kuo AG, Weiss C, Wood PL, O’Connor Hanson A, Kelso S, Harris RB, Disterhoft JF (2005) GLYX-13: a monoclonal antibody-derived peptide that acts as an N-methyl-D-aspartate receptor modulator. Neuropharmacology. 49:1077–1087CrossRefGoogle Scholar
  37. Murray EA, Bussey TJ, Saksida LM (2007) Visual perception and memory: a new view of medial temporal lobe function in primates and rodents. Annu Rev Neurosci 30:99–122CrossRefGoogle Scholar
  38. Nagayach A, Singh A, Geller AI (2019) Delivery of different genes into presynaptic and postsynaptic neocortical neurons connected by a BDNF-TrkB synapse. Brain Res 1712:16–24CrossRefGoogle Scholar
  39. Nakagawa S, Niimura Y, Gojobori T, Tanaka H, Miura K (2008) Diversity of preferred nucleotide sequences around the translation initiation codon in eukaryote genomes. Nucleic Acids Res 36:861–871CrossRefGoogle Scholar
  40. Niswender CM, Conn PJ (2010) Metabotropic glutamate receptors: physiology, pharmacology, and disease. Annu Rev Pharmacol Toxicol 50:295–322CrossRefGoogle Scholar
  41. Ohno K, Sawai K, lijima Y, Levin B, Meruelo D (1997) Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A. Nat Biotechnol 15:763–767CrossRefGoogle Scholar
  42. Osakada F, Mori T, Cetin AH, Marshel JH, Virgen B, Callaway EM (2011) New rabies virus variants for monitoring and manipulating activity and gene expression in defined neural circuits. Neuron. 71:617–631CrossRefGoogle Scholar
  43. Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates. Academic, SydneyGoogle Scholar
  44. Pop AS, Gomez-Mancilla B, Neri G, Willemsen R, Gasparini F (2014) Fragile X syndrome: a preclinical review on metabotropic glutamate receptor 5 (mGluR5) antagonists and drug development. Psychopharmacology 231:1217–1226CrossRefGoogle Scholar
  45. Rasmussen M, Kong L, Zhang GR, Liu M, Wang X, Szabo G, Curthoys NP, Geller AI (2007) Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter. Brain Res 1144:19–32CrossRefGoogle Scholar
  46. Ried MU, Girod A, Leike K, Buning H, Hallek M (2002) Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors. J Virol 76:4559–4566CrossRefGoogle Scholar
  47. Sengmany K, Gregory KJ (2016) Metabotropic glutamate receptor subtype 5: molecular pharmacology, allosteric modulation and stimulus bias. Br J Pharmacol 173:3001–3017CrossRefGoogle Scholar
  48. Smith IL, Hardwicke MA, Sandri-Goldin RM (1992) Evidence that the herpes simplex virus immediate early protein ICP27 acts post-transcriptionally during infection to regulate gene expression. Virology. 186:74–86CrossRefGoogle Scholar
  49. Song S, Wang Y, Bak SY, During MJ, Bryan J, Ashe O, Ullrey DB, Trask LE, Grant FD, O’Malley KL, Riedel H, Goldstein DS, Neve KA, LaHoste GJ, Marshall JF, Haycock JW, Neve RL, Geller AI (1998) Modulation of rat rotational behavior by direct gene transfer of constitutively active protein kinase C into nigrostriatal neurons. J Neurosci 18:4119–4132CrossRefGoogle Scholar
  50. Spear PG, Longnecker R (2003) Herpesvirus entry: an update. J Virol 77:10179–10185CrossRefGoogle Scholar
  51. Sun M, Zhang GR, Yang T, Yu L, Geller AI (1999) Improved titers for helper virus-free herpes simplex virus type 1 plasmid vectors by optimization of the packaging protocol and addition of noninfectious herpes simplex virus-related particles (previral DNA replication enveloped particles) to the packaging procedure. Hum Gene Ther 10:2005–2011CrossRefGoogle Scholar
  52. Tai CK, Logg CR, Park JM, Anderson WF, Press MF, Kasahara N (2003) Antibody-mediated targeting of replication-competent retroviral vectors. Hum Gene Ther 14:789–802CrossRefGoogle Scholar
  53. Volpers C, Thirion C, Biermann V, Hussmann S, Kewes H, Dunant P, von der Mark H, Herrmann A, Kochanek S, Lochmuller H (2003) Antibody-mediated targeting of an adenovirus vector modified to contain a synthetic immunoglobulin G-binding domain in the capsid. J Virol 77:2093–2104CrossRefGoogle Scholar
  54. Wang X, Kong L, Zhang GR, Sun M, Geller AI (2005) Targeted gene transfer to nigrostriatal neurons in the rat brain by helper virus-free HSV-1 vector particles that contain either a chimeric HSV-1 glycoprotein C--GDNF or a gC--BDNF protein. Molec Brain Res 139:88–102CrossRefGoogle Scholar
  55. Yang T, Zhang GR, Zhang W, Sun M, Wang X, Geller AI (2001) Enhanced reporter gene expression in the rat brain from helper virus-free HSV-1 vectors packaged in the presence of specific mutated HSV-1 proteins that affect the virion. Molec Brain Res 90:1–16CrossRefGoogle Scholar
  56. Zantomio D, Chana G, Laskaris L, Testa R, Everall I, Pantelis C, Skafidas E (2015) Convergent evidence for mGluR5 in synaptic and neuroinflammatory pathways implicated in ASD. Neurosci Biobehav Rev 52:172–177CrossRefGoogle Scholar
  57. Zhang G, Wang X, Yang T, Sun M, Zhang W, Wang Y, Geller AI (2000) A tyrosine hydroxylase–neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors. Molec Brain Res 84:17–31CrossRefGoogle Scholar
  58. Zhang G, Wang X, Kong L, Lu XG, Lee B, Liu M, Sun M, Franklin C, Cook RG, Geller AI (2005) Genetic enhancement of visual learning by activation of protein kinase C pathways in small groups of rat cortical neurons. J Neurosci 25:8468–8481CrossRefGoogle Scholar
  59. Zhang G, Cao H, Kong L, O'Brien J, Baughns A, Jan M, Zhao H, Wang X, Lu XG, Cook RG, Geller AI (2010a) Identified circuit in rat postrhinal cortex encodes essential information for performing specific visual shape discriminations. Proc Natl Acad Sci U S A 107:14478–14483CrossRefGoogle Scholar
  60. Zhang G, Cao H, Li X, Zhao H, Geller AI (2010b) Genetic labeling of both the axons of transduced, glutamatergic neurons in rat postrhinal cortex and their postsynaptic neurons in other neocortical areas by herpes simplex virus vectors that coexpress an axon-targeted ß-galactosidase and wheat germ agglutinin from a vesicular glutamate transporter-1 promoter. Brain Res 1361:1–11CrossRefGoogle Scholar
  61. Zhang G, Geller AI (2010) A helper virus-free HSV-1 vector containing the vesicular glutamate transporter-1 promoter supports expression preferentially in VGLUT1-containing glutamatergic neurons. Brain Res 1331:12–19CrossRefGoogle Scholar
  62. Zhang G, Zhao H, Cao H, Geller AI (2012a) Overexpression of either lysine-specific demethylase-1 or CLOCK, but not Co-Rest, improves long-term expression from a modified neurofilament promoter, in a helper virus-free HSV-1 vector system. Brain Res 1436:157–167CrossRefGoogle Scholar
  63. Zhang G, Zhao H, Cao H, Li X, Geller AI (2012b) Targeted gene transfer of different genes to presynaptic and postsynaptic neocortical neurons connected by a glutamatergic synapse. Brain Res 1473:173–184CrossRefGoogle Scholar
  64. Zhang G et al (2012c) CaMKII, MAPK, and CREB are coactivated in identified neurons in a cortical circuit required for performing visual shape discriminations. Hippocampus. 22:2276–2289CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of OphthalmologyLouisiana State University Health Sciences CenterNew OrleansUSA
  2. 2.Department of PharmacologyLouisiana State University Health Sciences CenterNew OrleansUSA

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