Neural circuits are exquisitely organized, consisting of many different neuronal subpopulations. However, it is difficult to assess the functional roles of these subpopulations using conventional extracellular recording techniques because these techniques do not easily distinguish spikes from different neuronal populations. To overcome this limitation, we have developed PINP (Photostimulation-assisted Identification of Neuronal Populations), a method of tagging neuronal populations for identification during in vivo electrophysiological recording. The method is based on expressing the light-activated channel channelrhodopsin-2 (ChR2) to restricted neuronal subpopulations. ChR2-tagged neurons can be detected electrophysiologically in vivo since illumination of these neurons with a brief flash of blue light triggers a short latency reliable action potential. We demonstrate the feasibility of this technique by expressing ChR2 in distinct populations of cortical neurons using two different strategies. First, we labeled a subpopulation of cortical neurons—mainly fast-spiking interneurons—by using adeno-associated virus (AAV) to deliver ChR2 in a transgenic mouse line in which the expression of Cre recombinase was driven by the parvalbumin promoter. Second, we labeled subpopulations of excitatory neurons in the rat auditory cortex with ChR2 based on projection target by using herpes simplex virus 1 (HSV1), which is efficiently taken up by axons and transported retrogradely; we find that this latter population responds to acoustic stimulation differently from unlabeled neurons. Tagging neurons is a novel application of ChR2, used in this case to monitor activity instead of manipulating it. PINP can be readily extended to other populations of genetically identifiable neurons, and will provide a useful method for probing the functional role of different neuronal populations in vivo.
References
[1]
Mountcastle VB, Talbot WH, Sakata H, Hyvarinen J (1969) Cortical neuronal mechanisms in flutter-vibration studied in unanesthetized monkeys. Neuronal periodicity and frequency discrimination. J Neurophysiol 32: 452–484.
[2]
Steriade M, Timofeev I, Durmuller N, Grenier F (1998) Dynamic properties of corticothalamic neurons and local cortical interneurons generating fast rhythmic (30–40 Hz) spike bursts. J Neurophysiol 79: 483–490.
[3]
Klausberger T, Magill PJ, Marton LF, Roberts JD, Cobden PM, et al. (2003) Brain-state- and cell-type-specific firing of hippocampal interneurons in vivo. Nature 421: 844–848.
[4]
Nowak LG, Azouz R, Sanchez-Vives MV, Gray CM, McCormick DA (2003) Electrophysiological classes of cat primary visual cortical neurons in vivo as revealed by quantitative analyses. J Neurophysiol 89: 1541–1566.
[5]
Mitchell JF, Sundberg KA, Reynolds JH (2007) Differential attention-dependent response modulation across cell classes in macaque visual area V4. Neuron 55: 131–141.
[6]
Evarts EV (1968) Relation of pyramidal tract activity to force exerted during voluntary movement. J Neurophysiol 31: 14–27.
[7]
Movshon JA, Newsome WT (1996) Visual response properties of striate cortical neurons projecting to area MT in macaque monkeys. J Neurosci 16: 7733–7741.
[8]
Hahnloser RH, Kozhevnikov AA, Fee MS (2002) An ultra-sparse code underlies the generation of neural sequences in a songbird. Nature 419: 65–70.
[9]
Swadlow HA, Gusev AG, Bezdudnaya T (2002) Activation of a cortical column by a thalamocortical impulse. J Neurosci 22: 7766–7773.
[10]
Kozhevnikov AA, Fee MS (2007) Singing-related activity of identified HVC neurons in the zebra finch. J Neurophysiol 97: 4271–4283.
[11]
Cauli B, Audinat E, Lambolez B, Angulo MC, Ropert N, et al. (1997) Molecular and physiological diversity of cortical nonpyramidal cells. J Neurosci 17: 3894–3906.
[12]
Margrie TW, Meyer AH, Caputi A, Monyer H, Hasan MT, et al. (2003) Targeted whole-cell recordings in the mammalian brain in vivo. Neuron 39: 911–918.
[13]
Nakai J, Ohkura M, Imoto K (2001) A high signal-to-noise Ca(2+) probe composed of a single green fluorescent protein. Nat Biotechnol 19: 137–141.
[14]
Miesenbock G (2004) Genetic methods for illuminating the function of neural circuits. Curr Opin Neurobiol 14: 395–402.
[15]
Mao T, O'Connor DH, Scheuss V, Nakai J, Svoboda K (2008) Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators. PLoS ONE 3: e1796.
[16]
Nagel G, Brauner M, Liewald JF, Adeishvili N, Bamberg E, et al. (2005) Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr Biol 15: 2279–2284.
[17]
Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8: 1263–1268.
[18]
Hippenmeyer S, Vrieseling E, Sigrist M, Portmann T, Laengle C, et al. (2005) A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol 3: e159.
[19]
Kuhlman SJ, Huang ZJ (2008) High-resolution labeling and functional manipulation of specific neuron types in mouse brain by Cre-activated viral gene expression. PLoS ONE 3: e2005.
[20]
Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci U S A 85: 5166–5170.
[21]
Lilley CE, Groutsi F, Han Z, Palmer JA, Anderson PN, et al. (2001) Multiple immediate-early gene-deficient herpes simplex virus vectors allowing efficient gene delivery to neurons in culture and widespread gene delivery to the central nervous system in vivo. J Virol 75: 4343–4356.
[22]
Games KD, Winer JA (1988) Layer V in rat auditory cortex: projections to the inferior colliculus and contralateral cortex. Hear Res 34: 1–25.
[23]
Budinger E, Heil P, Hess A, Scheich H (2006) Multisensory processing via early cortical stages: Connections of the primary auditory cortical field with other sensory systems. Neuroscience 143: 1065–1083.
[24]
Henze DA, Borhegyi Z, Csicsvari J, Mamiya A, Harris KD, et al. (2000) Intracellular features predicted by extracellular recordings in the hippocampus in vivo. J Neurophysiol 84: 390–400.
[25]
Hromadka T, Deweese MR, Zador AM (2008) Sparse representation of sounds in the unanesthetized auditory cortex. PLoS Biol 6: e16.
[26]
Chung S, Li X, Nelson SB (2002) Short-term depression at thalamocortical synapses contributes to rapid adaptation of cortical sensory responses in vivo. Neuron 34: 437–446.
[27]
Wehr M, Zador AM (2005) Synaptic mechanisms of forward suppression in rat auditory cortex. Neuron 47: 437–445.
[28]
Otazu GH, Tai LH, Yang Y, Zador AM (2009) Engaging in an auditory task suppresses responses in auditory cortex. Nat Neurosci 12: 646–654.
[29]
Deisseroth K, Feng G, Majewska AK, Miesenbock G, Ting A, et al. (2006) Next-generation optical technologies for illuminating genetically targeted brain circuits. J Neurosci 26: 10380–10386.
[30]
Zhang F, Wang LP, Boyden ES, Deisseroth K (2006) Channelrhodopsin-2 and optical control of excitable cells. Nat Methods 3: 785–792.
[31]
Zemelman BV, Lee GA, Ng M, Miesenbock G (2002) Selective photostimulation of genetically chARGed neurons. Neuron 33: 15–22.
[32]
Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7: 1381–1386.
[33]
Lima SQ, Miesenbock G (2005) Remote control of behavior through genetically targeted photostimulation of neurons. Cell 121: 141–152.
[34]
Schroll C, Riemensperger T, Bucher D, Ehmer J, Voller T, et al. (2006) Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae. Curr Biol 16: 1741–1747.
[35]
Atasoy D, Aponte Y, Su HH, Sternson SM (2008) A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J Neurosci 28: 7025–7030.
[36]
Arenkiel BR, Peca J, Davison IG, Feliciano C, Deisseroth K, et al. (2007) In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54: 205–218.
[37]
Petreanu L, Huber D, Sobczyk A, Svoboda K (2007) Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections. Nat Neurosci 10: 663–668.
[38]
Zhang YP, Holbro N, Oertner TG (2008) Optical induction of plasticity at single synapses reveals input-specific accumulation of alphaCaMKII. Proc Natl Acad Sci U S A 105: 12039–12044.
[39]
Dittgen T, Nimmerjahn A, Komai S, Licznerski P, Waters J, et al. (2004) Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci U S A 101: 18206–18211.
[40]
Jung JC, Mehta AD, Aksay E, Stepnoski R, Schnitzer MJ (2004) In vivo mammalian brain imaging using one- and two-photon fluorescence microendoscopy. J Neurophysiol 92: 3121–3133.
[41]
Lin JY, Lin MZ, Steinbach P, Tsien RY (2009) Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96: 1803–1814.
[42]
McCormick DA, Connors BW, Lighthall JW, Prince DA (1985) Comparative electrophysiology of pyramidal and sparsely spiny stellate neurons of the neocortex. J Neurophysiol 54: 782–806.
[43]
Wu GK, Arbuckle R, Liu BH, Tao HW, Zhang LI (2008) Lateral sharpening of cortical frequency tuning by approximately balanced inhibition. Neuron 58: 132–143.
[44]
Hefti BJ, Smith PH (2000) Anatomy, physiology, and synaptic responses of rat layer V auditory cortical cells and effects of intracellular GABA(A) blockade. J Neurophysiol 83: 2626–2638.
[45]
Turner JG, Hughes LF, Caspary DM (2005) Affects of aging on receptive fields in rat primary auditory cortex layer V neurons. J Neurophysiol 94: 2738–2747.
[46]
Preston CM (2000) Repression of viral transcription during herpes simplex virus latency. J Gen Virol 81: 1–19.
