Injection of Adeno-Associated Virus Containing Optogenetic and Chemogenetic Probes into the Neonatal Mouse Brain

  • Nhan C. Huynh
  • Baher A. Ibrahim
  • Christopher M. Lee
  • Mickeal N. Key
  • Daniel A. LlanoEmail author
Part of the Neuromethods book series (NM, volume 152)


Optogenetics and chemogenetics are neuromodulation techniques used to study neuronal pathways. Viral vectors containing optogenetic or chemogenetic probes require at least a week after injection to produce effective expression levels in neurons. Therefore, injections need to be done in neonatal mice to enable experiments that require mice younger than 30 days of age. Here, we describe a protocol for performing surgery on neonatal mice and brain injection of a viral vector. The procedure utilizes cryoanesthesia and a pressure injector with a micropipette that can be directly injected through the skull of neonatal mice. Compared to other approaches, this protocol is relatively easy to implement and takes only minutes to perform, additionally allowing for increased numbers of injections. In the examples shown in this chapter, viral vectors were successfully delivered into the auditory cortex and the hippocampus as indicated by labeled expression of the optogenetic/chemogenetic probes. This technique provides a method for investigators to perform surgical injections and optogenetic/chemogenetic experiments in neonatal mice.

Key words

Optogenetics Chemogenetics Surgery Rodent Neonatal mouse Cryoanesthesia Adeno-associated virus 



The research was supported by research grant DC013073 from the National Institutes of Health.


  1. 1.
    Mattis J, Tye KM, Ferenczi EA, Ramakrishnan C, O’Shea DJ, Prakash R, Gunaydin LA, Hyun M, Fenno LE, Gradinaru V, Yizhar O, Deisseroth D (2012) Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nat Methods 9(2):159–172CrossRefGoogle Scholar
  2. 2.
    Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8(9):1263–1268CrossRefGoogle Scholar
  3. 3.
    Zhang F, Wang LP, Brauner M, Liewald JF, Kay K, Watzke N, Wood PG, Bamberg E, Nagel G, Gottschalk A, Deisseroth K (2007) Multimodal fast optical interrogation of neural circuitry. Nature 446(7136):633–639CrossRefGoogle Scholar
  4. 4.
    Klapoetke NC, Murata Y, Kim SS, Pulver SR, Birdsey-Benson A, Cho YK, Moriomoto TK, Chuong AS, Carpenter EJ, Tian Z, Wang J, Xie Y, Yan Z, Zhang Y, Chow BY, Surek B, Melkonian M, Jayaraman V, Constantine-Paton M, Wong GK, Boyden ES (2014) Independent optical excitation of distinct neural populations. Nat Methods 11(3):338–346CrossRefGoogle Scholar
  5. 5.
    Cardin JA, Carlén M, Meletis K, Knoblich U, Zhang F, Deisseroth K, Tsai L, Moore CI (2010) Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2. Nat Protoc 5(2):247–254CrossRefGoogle Scholar
  6. 6.
    Beltramo R, D’Urso G, Dal Maschio M, Farisello P, Bovetti S, Clovis Y, Lassi G, Tucci V, Tonelli DDP, Fellin T (2013) Layer-specific excitatory circuits differentially control recurrent network dynamics in the neocortex. Nat Neurosci 16(2):227–234CrossRefGoogle Scholar
  7. 7.
    Roth BL (2016) DREADDs for neuroscientists. Neuron 89(4):683–694CrossRefGoogle Scholar
  8. 8.
    Armbruster BN, Li X, Pausch MH, Herlitze S, Roth BL (2007) Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc Natl Acad Sci 104(12):5163–5168CrossRefGoogle Scholar
  9. 9.
    Armbruster B, Roth B (2005) Creation of designer biogenic amine receptors via directed molecular evolution. In: Neuropsychopharmacology, vol 30. Nature Publishing Group, London, pp S265–S265Google Scholar
  10. 10.
    Alexander GM, Rogan SC, Abbas AI, Armbruster BN, Pei Y, Allen JA, Nonneman RJ, Hartmann J, Moy SS, Nicolelis MA, McNamara JO, Roth BL (2009) Remote control of neuronal activity in transgenic mice expressing evolved G protein-coupled receptors. Neuron 63(1):27–39CrossRefGoogle Scholar
  11. 11.
    Vardy E, Robinson JE, Li C, Olsen RH, DiBerto JF, Giguere PM, Sassano FM, Huang X, Zhu H, Urban DJ, White KL, Rittiner JE, Crowley NA, Pleil KE, Mazzone CM, Mosier PD, Song J, Kash TL, Malanga CJ, Krashes MJ, Roth BL (2015) A new DREADD facilitates the multiplexed chemogenetic interrogation of behavior. Neuron 86(4):936–946CrossRefGoogle Scholar
  12. 12.
    Stachniak TJ, Ghosh A, Sternson SM (2014) Chemogenetic synaptic silencing of neural circuits localizes a hypothalamus → midbrain pathway for feeding behavior. Neuron 82(4):797–808CrossRefGoogle Scholar
  13. 13.
    Allen JA, Roth BL (2011) Strategies to discover unexpected targets for drugs active at G protein–coupled receptors. Annu Rev Pharmacol Toxicol 51:117–144CrossRefGoogle Scholar
  14. 14.
    Ferguson SM, Phillips PE, Roth BL, Wess J, Neumaier JF (2013) Direct-pathway striatal neurons regulate the retention of decision-making strategies. J Neurosci 33(28):11668–11676CrossRefGoogle Scholar
  15. 15.
    Roth BL, Craigo SC, Choudhary MS, Uluer A, Monsma FJ, Shen Y et al (1994) Binding of typical and atypical antipsychotic agents to 5-hydroxytryptamine-6 and 5-hydroxytryptamine-7 receptors. J Pharmacol Exp Ther 268(3):1403–1410PubMedGoogle Scholar
  16. 16.
    Chavkin C, Sud S, Jin W, Stewart J, Zjawiony JK, Siebert DJ, Toth BA, Hufeisen SJ, Roth BL (2004) Salvinorin A, an active component of the hallucinogenic sage Salvia divinorum is a highly efficacious κ-opioid receptor agonist: structural and functional considerations. J Pharmacol Exp Ther 308(3):1197–1203CrossRefGoogle Scholar
  17. 17.
    Jann MW, Lam YW, Chang WH (1994) Rapid formation of clozapine in guinea-pigs and man following clozapine-N-oxide administration. Archiv Int Pharmacodyn Ther 328(2):243–250Google Scholar
  18. 18.
    Chen X, Choo H, Huang XP, Yang X, Stone O, Roth BL, Jin J (2015) The first structure–activity relationship studies for designer receptors exclusively activated by designer drugs. ACS Chem Nerosci 6(3):476–484CrossRefGoogle Scholar
  19. 19.
    Zhu H, Pleil KE, Urban DJ, Moy SS, Kash TL, Roth BL (2014) Chemogenetic inactivation of ventral hippocampal glutamatergic neurons disrupts consolidation of contextual fear memory. Neuropsychopharmacology 39(8):1880–1892CrossRefGoogle Scholar
  20. 20.
    Scofield MD, Boger HA, Smith RJ, Li H, Haydon PG, Kalivas PW (2015) Gq-DREADD selectively initiates glial glutamate release and inhibits cue-induced cocaine seeking. Biol Psychiatry 78(7):441–451CrossRefGoogle Scholar
  21. 21.
    Peñagarikano O, Lázaro MT, Lu XH, Gordon A, Dong H, Lam HA, Peles E, Maidment NT, Murphy NP, Yang XW, Golshani P, Geschwind DH (2015) Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism. Sci Transl Med 7(271):271ra8–271ra8CrossRefGoogle Scholar
  22. 22.
    Roth BL, Marshall FH (2012) NOBEL 2012 chemistry: studies of a ubiquitous receptor family. Nature 492(7427):57–57CrossRefGoogle Scholar
  23. 23.
    Samama P, Cotecchia S, Costa T, Lefkowitz RJ (1993) A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem 268(7):4625–4636PubMedGoogle Scholar
  24. 24.
    Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher DS, Maratos-Flier E, Roth BL, Lowell BB (2011) Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J Clin Invest 121(4):1424CrossRefGoogle Scholar
  25. 25.
    Phifer CB, Terry LM (1986) Use of hypothermia for general anesthesia in preweanling rodents. Physiol Behav 38(6):887–890CrossRefGoogle Scholar
  26. 26.
    National Research Council (2003) Guidelines for the care and use of mammals in neuroscience and behavioral research. National Academies Press, Washington, DCGoogle Scholar
  27. 27.
    Kim JY, Grunke SD, Levites Y, Golde TE, Jankowsky JL (2013) Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J Vis Exp (91):51863–51863Google Scholar
  28. 28.
    Watson DJ, Passini MA, Wolfe JH (2005) Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors. Hum Gene Ther 16(1):49–56CrossRefGoogle Scholar
  29. 29.
    Chakrabarty P, Rosario A, Cruz P, Siemienski Z, Ceballos-Diaz C, Crosby K, Jansen K, Borchelt DR, Kim J, Jankowsky JL, Golde TE (2013) Capsid serotype and timing of injection determines AAV transduction in the neonatal mice brain. PLoS One 8(6):e67680CrossRefGoogle Scholar
  30. 30.
    Ibrahim BA, Wang H, Lesicko AM, Bucci B, Paul K, Llano DA (2017) Effect of temperature on FAD and NADH-derived signals and neurometabolic coupling in the mouse auditory and motor cortex. Pflügers Arch 469(12):1631–1649CrossRefGoogle Scholar
  31. 31.
    Cruikshank SJ, Rose HJ, Metherate R (2002) Auditory thalamocortical synaptic transmission in vitro. J Neurophysiol 87(1):361–384CrossRefGoogle Scholar
  32. 32.
    Stebbings KA, Choi HW, Ravindra A, Caspary DM, Turner JG, Llano DA (2016) Ageing-related changes in GABAergic inhibition in mouse auditory cortex, measured using in vitro flavoprotein autofluorescence imaging. J Physiol 594(1):207–221CrossRefGoogle Scholar
  33. 33.
    Optogenetics Material Request/FAQ (n.d.) Optogenetics Resource Center.
  34. 34.
    Davis HE, Morgan JR, Yarmush ML (2002) Polybrene increases retrovirus gene transfer efficiency by enhancing receptor-independent virus adsorption on target cell membranes. Biophys Chem 97(2):159–172CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Nhan C. Huynh
    • 1
    • 2
    • 3
  • Baher A. Ibrahim
    • 1
    • 2
  • Christopher M. Lee
    • 1
    • 2
  • Mickeal N. Key
    • 2
    • 4
  • Daniel A. Llano
    • 1
    • 2
    • 4
    Email author
  1. 1.Department of Molecular and Integrative PhysiologyUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Beckman Institute for Advanced Science and TechnologyUrbanaUSA
  3. 3.Massachusetts Institute of TechnologyCambridgeUSA
  4. 4.Neuroscience ProgramUniversity of Illinois at Urbana-ChampaignUrbanaUSA

Personalised recommendations