Transfer of optogenetic vectors into the brain of neonatal animals to study neuron functions during subsequent periods of development

Optogenetics, that is, the control of cell activity using light-sensitive ion channels opsins with light of a specific wavelength, is increasingly being used to study activities and functions of neurons. Expression of opsins in the cell membrane, followed by the acquisition by the cell of the sensitivity to light is achieved by means of viral vectors, often created on the basis of lentiviral or adeno-associated (AAV) viruses bearing the nucleotide sequence encoding the photo-channel proteins. Inclusion of the cell-specific promoter of interest into the transgene-expression cassette allows opsin to be produced only in the target cells. The aim of this work was to briefly describe the optogenetic method, as well as to analyze the possibility to use administration of viral vectors into the brain of neonatal animals to study the function of neurons in vivo during subsequent periods of development. In this analysis, 3-day-old rat pups received intracerebroventricular injections of optovector (pAAV-CAMKIIa-ChR2h134-YFP), coding for a photo channel, which activates neurons, and the yellow fluorescent marker protein under the CAMKIIa promoter specific for glutamatergic neurons under cold anesthesia. The peak expression of the transferred gene is usually achieved at week 3–5 after the transfer of the vector, which is what was also observed in our experiments. Stimulation of the hippocampal neurons with blue light in the 20-day-old animals, to which opto-vector was transferred at the 3rd day of life, increased the discharge activity of these neurons. This light stimulation increased expression of the recognized marker of neuronal activation protein c-Fos in these photosensitive cells too. The same experiments with older animals, 60 days after the neonatal opto-channel gene transfer, revealed no noticeable expression of this channel or photoactivation of target neurons of the hippocampus. Thus, neonatal administration of a viral vector carrying an opto-channel gene is suitable for the study of brain neurons in rats of juvenile age, and requires additional control for gene expression during subsequent periods of development.

1. Ambrosi C.M., Boyle P.M., Chen K., Trayanova N.A., Entcheva E. Optogeneticsenabled assessment of viral gene and cell therapy for restoration of cardiac excitability. Sci. Rep. 2015;5(17350). DOI 10.1038/srep17350

2. Arenkiel B.R., Peca J., Davison I.G., Feliciano C., Deisseroth K., Augustine G.J., Ehlers M.D., Feng G. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron. 2007;54(2):205-218. DOI 10.1016/j.neuron.2007.03.005

3. Boyden E.S., Zhang F., Bamberg E., Nagel G., Deisseroth K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 2005;8(9):1263-1268. DOI 10.1038/nn1525

4. Buzsaki G., Stark E., Berenyi A., Khodagholy D., Kipke D.R., Yoon E., Wise K.D. Tools for probing local circuits: high-density silicon probes combined with optogenetics. Neuron. 2015;86(1):92-105. DOI 10.1016/j.neuron.2015.01.028

5. Davis B.J., Marks D.L., Ralston T.S., Carney P.S., Boppart S.A. Interferometric synthetic aperture microscopy: Computed imaging for scanned coherent microscopy. Sensors (Basel). 2008;8(6):3903-3931. DOI 10.3390/s8063903

6. Deisseroth K. Optogenetics. Nat. Methods. 2011;8(1):26-29. DOI 10.1038/nmeth.f.324

7. Diester I., Kaufman M.T., Mogri M., Pashaie R., Goo W., Yizhar O., Ramakrishnan C., Deisseroth K., Shenoy K.V. An optogenetic toolbox designed for primates. Nat. Neurosci. 2011;14(3):387-397. DOI 10.1038/nn.2749

8. Dittgen T., Nimmerjahn A., Komai S., Licznerski P., Waters J., Margrie T.W., Helmchen F., Denk W., Brecht M., Osten P. Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc. Natl Acad. Sci. USA. 2004;101(52):18206-18211. DOI 10.1073/pnas.0407976101

9. Dygalo N.N. Optogenetics in Investigations of Brain Mechanisms of Behavior. Uspekhi fiziologicheskikh nauk = Advance in Physiological Sciences (Moscow). 2015;46(2):17- 23.

10. Dygalo N.N., Kalinina T.S., Shishkina G.T. Neonatal programming of rat behavior by downregulation of alpha2A-adrenoreceptor gene expression in the brain. Ann. N.Y. Acad. Sci. 2008;1148:409-414. DOI 10.1196/annals.1410.063

11. Glock C., Nagpal J., Gottschalk A. Microbial rhodopsin optogenetic tools: Application for analyses of synaptic transmission and of neuronal network activity in behavior. Meth. Mol. Biol. 2015;1327:87-103. DOI 10.1007/978-1-4939-2842-2_8

12. Gompf H.S., Budygin E.A., Fuller P.M., Bass C.E. Targeted genetic manipulations of neuronal subtypes using promoter-specific combinatorial AAVs in wild-type animals. Front. Behav. Neurosci. 2015;9:152. DOI 10.3389/fnbeh.2015.00152

13. Gradinaru V., Thompson K.R., Zhang F., Mogri M., Kay K., Schneider M.B., Deisseroth K. Targeting and readout strategies for fast optical neural control in vitro and in vivo. J. Neurosci. 2007;27(52): 14231-14238. DOI 10.1523/JNEUROSCI.3578-07.2007

14. Guenthner C.J., Miyamichi K., Yang H.H., Heller H.C., Luo L. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations. Neuron. 2013;78(5):773-784. DOI 10.1016/j.neuron.2013.03.025

15. Hioki H., Kameda H., Nakamura H., Okunomiya T., Ohira K., Nakamura K., Kuroda M., Furuta T., Kaneko T. Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene Ther. 2007;14(11):872-882. DOI 10.1038/sj.gt.3302924

16. Husson S.J., Liewald J.F., Schultheis C., Stirman J.N., Lu H., Gottschalk A. Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans. PloS One. 2012;7(7):e40937. DOI 10.1371/journal.pone.0040937

17. Kim J.Y., Ash R.T., Ceballos-Diaz C., Levites Y., Golde T.E., Smirnakis S.M., Jankowsky J.L. Viral transduction of the neonatal brain delivers controllable genetic mosaicism for visualising and manipulating neuronal circuits in vivo. Eur. J. Neurosci. 2013;37(8):1203-1220. DOI 10.1111/ejn.12126

18. Lanshakov D.A., Sukhareva E.V., Kalinina T.S., Dygalo N.N. Dexamethasone-induced acute excitotoxic cell death in the developing brain. Neurobiol. Dis. 2016;91:1-9 DOI 10.1016/j.nbd.2016.02.009

19. Lin J.Y. A user’s guide to channelrhodopsin variants: features, limitations and future developments. Exp. Physiol. 2011;96(1):19-25. DOI 10.1113/expphysiol.2009.051961

20. Liu X., Ramirez S., Pang P.T., Puryear C.B., Govindarajan A., Deisseroth K., Tonegawa S. Optogenetic stimulation of a hippocampal engram activates fear memory recall. Nature. 2012;484(7394):381- 385. DOI 10.1038/nature11028

21. Pisanello F., Sileo L., Oldenburg I.A., Pisanello M., Martiradonna L., Assad J.A., Sabatini B.L., De Vittorio M. Multipoint-emitting optical fibers for spatially addressable in vivo optogenetics. Neuron. 2014;82(6):1245-1254. DOI 10.1016/j.neuron.2014.04.041

22. Samaranch L., San Sebastian W., Kells A.P., Salegio E.A., Heller G., Bringas J.R., Pivirotto P., DeArmond S., Forsayeth J., Bankiewicz K.S. AAV9-mediated expression of a non-self protein in nonhuman primate central nervous system triggers widespread neuroinflammation driven by antigen-presenting cell transduction. Mol. Ther. 2014;22(2):329-337. DOI 10.1038/mt.2013.266

23. Shishkina G.T., Kalinina T.S., Dygalo N.N. Attenuation of alpha2Aadrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood. Neuroscience. 2004;129(3):521-528. DOI 10.1016/j.neuroscience.2004.08.015

24. Shishkina G.T., Kalinina T.S., Sournina N.Y., Dygalo N.N. Effects of antisense to the (alpha)2A-adrenoceptors administered into the region of the locus ceruleus on behaviors in plus-maze and sexual behavior tests in sham-operated and castrated male rats. J. Neurosci. 2001;21(2):726-731.

25. Stuber G.D., Sparta D.R., Stamatakis A.M., van Leeuwen W.A., Hardjoprajitno J.E., Cho S., Tye K.M., Kempadoo K.A., Zhang F., Deisseroth K., Bonci A. Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking. Nature. 2011;475(7356):377-380. DOI 10.1038/nature10194

26. Tan E.L., Pereles B.D., Horton B., Shao R., Zourob M., Ong K.G. Implantable biosensors for real-time strain and pressure monitoring. Sensors (Basel). 2008;8(10):6396-6406. DOI 10.3390/s8106396

27. Wang H., Peca J., Matsuzaki M., Matsuzaki K., Noguchi J., Qiu L., Wang D., Zhang F., Boyden E., Deisseroth K., Kasai H., Hall W.C., Feng G., Augustine G.J. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl Acad. Sci. USA. 2007;104(19):8143-8148. DOI 10.1073/pnas.0700384104

28. Warden M.R., Selimbeyoglu A., Mirzabekov J.J., Lo M., Thompson K.R., Kim S.Y., Adhikari A., Tye K.M., Frank L.M., Deisseroth K. A prefrontal cortex-brainstem neuronal projection that controls response to behavioural challenge. Nature. 2012;492(7429):428-432.DOI 10.1038/nature11617

29. Watakabe A., Ohtsuka M., Kinoshita M., Takaji M., Isa K., Mizukami H., Ozawa K., Isa T., Yamamori T. Comparative analyses of adeno-associated viral vector serotypes 1, 2, 5, 8 and 9 in marmoset, mouse and macaque cerebral cortex. Neurosci. Res. 2015;93:144-157. DOI 10.1016/j.neures.2014.09.002

30. Zapara G.A., Ratushnyak A.S., Shtark M.B. Local changes in transmembrane ionic currents during plastic reorganizations of electrogenesis of isolated neurons of the pond snail. Neurosci. Behav. Physiol. 1989;19(3):224-229.

31. Zhang F., Gradinaru V., Adamantidis A.R., Durand R., Airan R.D., de Lecea L., Deisseroth K. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nat. Protoc. 2010;5(3):439-456. DOI 10.1038/nprot.2009.226

32. Zhang F., Wang L.P., Brauner M., Liewald J.F., Kay K., Watzke N., Wood P.G., Bamberg E., Nagel G., Gottschalk A., Deisseroth K. Multimodal fast optical interrogation of neural circuitry. Nature. 2007;446(7136):633-639. DOI 10.1038/nature05744

33. Zhao S., Ting J.T., Atallah H.E., Qiu L., Tan J., Gloss B., Augustine G.J., Deisseroth K., Luo M., Graybiel A.M., Feng G. Cell typespecific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function. Nat. Meth. 2011;8(9):745-752.