Transgenic expression of optical actuators
Individual opsin molecules induce relatively small changes in membrane polarization following photostimulation. Because of this, initial attempts to develop mice that express effective levels of the optical activating molecule, channelrhodopsin-2, in specific neuronal cell populations were based on directly linking the ChR2 gene to very strong cell type-specific promoters, such as those from Thy1
(Arenkiel et al., 2007
; Wang et al., 2007
(Hagglund et al., 2010
(Dhawale et al., 2010
) and Chat
(Ren et al., 2011
), or to the Tet-inducible promoter (Chuhma et al., 2011
). Characterization of these mice revealed that membrane depolarization and spiking activity were evoked in predicted cell types following blue light stimulation. In contrast, expression of optical inhibitory molecules in mice has been more problematic, mainly due to protein aggregation and low current conductance (Chuhma et al., 2011
; Zhao et al., 2008
). To date, there has been only one report of functional NpHR expression in transgenic mice, using the Orexin promoter (Tsunematsu et al., 2011
). Efforts to overcome limitations associated with early versions of the silencing opsins have led to the development of second- and third-generation optical silencing molecules, including eNpHR (Gradinaru et al., 2008
), eNpHR3.0 (Gradinaru et al., 2010
), various forms of Arch (Chow et al., 2010
), and ArchT (Han et al., 2011
). With greatly improved membrane expression and photoconductance, these reengineered opsins embody a major step toward reliable genetic silencing. Below we provide an overview of the published transgenic mouse lines expressing optical activators or silencers (see for a summary).
Transgenic mouse lines expressing optical activators or silencers
The first ChR2 transgenic mouse line was made using the Thy1
promoter, which had been shown to enable extremely high-level brain expression (Caroni, 1997
; Feng et al., 2000
), and it demonstrated for the first time the in vivo
potential of ChR2 to investigate neural circuit properties (Arenkiel et al., 2007
; Wang et al., 2007
). The Thy1
-ChR2-EYFP construct was randomly integrated into the mouse genome via pronuclear injection, and several transgenic lines with differential levels and patterns of expression were described. In the cortical pyramidal neurons of line 18 mice (Wang et al., 2007
), which showed the highest CNS expression of ChR2, maximal peak photocurrents of 500–600 pA were reached with large-area-applied blue light pulses (~10 mW/mm2
of 5–10 ms duration). Action potentials could be induced by light as low as 0.2 mW/mm2
, with ~6 ms average latency from light onset. Action potentials fired reliably following light pulses up to 30 Hz. These photoexcitation properties are similar to those of ChR2-positive neurons generated by viral transduction or in utero
electorportion. In line 9 mice, a two-dimensional array light scanning method over the surface of a cell was used to map local synaptic inputs to ChR2-negative cells. Mapping data revealed that excitatory synaptic input maps to pyramidal neurons were qualitatively different from those to interneurons, with the former being much larger and more irregularly shaped. Furthermore, it was demonstrated for the first time that ChR2-positive cortical layer 5 cells can be activated in vivo
by light on a millisecond scale (Arenkiel et al., 2007
). Brief light pulses (3.5 ms), delivered through a 200-µm diameter optical fiber positioned directly above the surface of the brain, reliably evoked responses from individual neurons with an average of ~10 ms spike latency. Neurons followed spike trains at high fidelity at frequencies as high as 40 Hz.
In the olfactory bulb of line 18 Thy1
-ChR2-EYFP mice (Arenkiel et al., 2007
), only mitral cells have ChR2 expression, and in these cells photocurrents (mean of ~200 pA) and action potentials could be reliably induced by blue light in vitro
. Illuminating the dorsal surface of the olfactory bulb could induce spiking in mitral cells. A circuit mapping study was conducted by stimulating mitral cells in the bulb and recording postsynaptic responses in piriform cortex. Large area (600 µm) light stimuli on the bulb drove the firing of piriform neurons much more effectively than smaller area (300 µm and 100 µm) light stimuli, supporting the model that multiple distant mitral cells or glomerular inputs are required for the activation of piriform cortical cells.
The 12-kb Omp
promoter, another strong neuronal promoter, drives expression in all olfactory sensory neurons (OSNs), and several Omp
-ChR2-EYFP transgenic mouse lines have been generated by pronuclear injection (Dhawale et al., 2010
). Two transgenic lines had ChR2-EYFP expression restricted to the vomeronasal organ and the accessory olfactory bulb. A third line (termed ORC-M) that expressed ChR2-EYFP in the olfactory epithelium and the main olfactory bulb, as well as in the vomeronasal organ and accessory olfactory bulb, was used for study. Light stimulation in the glomerular layer activated ChR2 in the OSN axon terminals, which led to glutamate release and generated postsynaptic currents in the mitral cells. Light stimulation over the olfactory bulb surface induced a rapid and reliable increase of firing of the mitral/tufted cells through presynaptic activation (as opposed to the direct activation observed using cells from the Thy1
-ChR2-EYFP mice above). Titration of light intensity (to lower than 2 mW/mm2
) enabled mapping of a single glomerulus to each recorded single-unit mitral or tufted cell. Tetrad recordings coupled with light stimulation identified neighboring mitral/tufted cell pairs that are either sister cells (i.e., cells receiving inputs from the same glomerulus) or non-sister cells (i.e., cells receiving inputs from different glomeruli). It was found that sister mitral cells showed correlated changes in their firing rate, whereas non-sister cells did not. Intriguingly, however, odor presentation desynchronized sister cells, suggesting that the odor response properties of sister cells are not redundant and could be differentially affected by local circuitry.
-ChR2-YFP transgenic mouse line was generated using a BAC transgenic strategy in which human codon-optimized hChR2-YFP was inserted in frame at the start codon of Vglut2
(also known as Slc17a6
) in the BAC (Hagglund et al., 2010
). In these mice ChR2 is specifically expressed in Vglut2
expressing glutamatergic neurons, including those in the hindbrain and spinal cord, with variable intensities. Spinal cord ChR2-positive neurons responded to continuous blue light (~35 mW/mm2
) with rapid depolarization of variable degrees (1–15 mV). The relatively small and variable depolarization might be related to the low and variable expression of ChR2 in these transgenic mice. Nonetheless, light stimulation of the glutamatergic neurons in the spinal cord or the hindbrain from the ventral side was sufficient to generate rhythmic locomotor-like activities, demonstrating the direct involvement of these glutamatergic neurons in intrinsic rhythm generation.
-ChR2-EYFP mouse line was also generated using the BAC transgenic strategy (Ren et al., 2011
). It was used to study the cholinergic transmission in the habenulo-interpeduncular pathway. ChR2-EYFP fluorescence was found in the ventral two-thirds of the medial habenula (MHb) where CHAT+ cells are located, as well as the entire MHb-fr-IPN axon projection tract. Brief (5 ms) or continuous blue light pulses (20 mW/mm2
) could evoke rapid action potential firing and large photocurrents (~500 pA peak amplitude) in MHb neurons. Light stimulation of the axonal terminals in the projection target area, interpeduncular nucleus (IPN), elicited postsynaptic responses in IPN neurons tested. Brief (5 ms) light pulses evoked fast EPSCs that were blocked only by glutamate antagonists. Prolonged light stimulation evoked an additional component, the slow inward currents that were not affected by glutamate antagonists but were reduced by nAchR blockers to about one-third of their original levels, thus revealing the dual transmission nature of the MHb cholinergic neurons.
-ChR2-Venus knock-in mice were generated by inserting the mammalian codon-optimized ChR2(H134R)-Venus gene into the Mrgprd
gene in frame at the start codon through homologous recombination (Wang and Zylka, 2009
molecularly marks ~75% of all IB4+ nonpeptidergic nociceptive neurons in the dorsal root ganglia (DRGs) and trigeminal ganglia, and these Mrgprd+ neurons exclusively innervate skin and terminate in lamina II (the substantia gelatinosa or SG) of dorsal spinal cord. Light stimulation evoked action potentials in 68% heterogyzous and 94% homozygous Mrgprd
-ChR2-Venus+ DRG cells in dissociated culture. The latency between light onset and actional potential peak was ~20 ms, and the spike jitter (average standard deviation of the light-evoked spike latency from each neuron) was ~1.3 ms. In spinal cord slices, light-evoked excitatory postsynaptic currents (EPSCL) could be generated in ~50% of SG neurons. Using spike jitter and pre-post synaptic proximity as criteria, the connections between the Mrgprd
-ChR2-Venus+ DRG cells and these light responsive SG neurons were classified into monosynaptic or polysynaptic. And it was found that monosynaptic connections made up of ~50% of the light responsive SG cells and included almost all known SG cell types except for the islet cells.
Conditional expression of ChR2 using the Tet system was achieved in the BTR mice (Chuhma et al., 2011
), in which a bi-directional tetO (also known as TRE) promoter was used to drive ChR2-mCherry in one direction and HaloR-EGFP (HaloR being the same as NpHR) in the other direction. After pronuclear injection, 3 founder lines were obtain and crossed to a line of αCaMKII-tTA with striatal tTA expression restricted to medium spiny neurons (MSNs). Only one of the 3 crosses (αCaMKIIa-tTA::BTR6) had ChR2-mCherry expression, and was subsequently used in the study. Within the dorsal striatum, <10% MSNs were fluorescently labeled and they were randomly distributed in a mosaic pattern. While mCherry fluorescence was seen in the membrane, outlining the cell soma, and extended into the processes, EGFP accumulated in aggresomes in MSN cell bodies and was not seen in the plasma membrane, suggesting that ChR2, but not HaloR, would be functional. While yellow light illumination elicited marginal HaloR responses, blue light illumination evoked large ChR2 photocurrents (~350 pA) and depolarization (~30 mV). ChR2 activation was therefore used to map functional connections among different cell types in both specificity and strength (as measured in the size of IPSCs). Photostimulation of MSN presynaptic terminals expressing ChR2 elicits GABAA
synaptic responses from local collaterals in the dorsal striatum (dStr), as well as in their projections to globus pallidus (GP) and substantia nigra (SN). It was found that MSN synaptic connections are quite specific. In the dStr, MSNs connect robustly to other MSNs and less robustly to tonically active neurons, but not to fast-spiking interneurons. In the GP, MSNs connect strongly to type B/C neurons and practically not to type A neurons. In the SN, MSNs make their strongest connections with SNr GABAergic neurons, but no connections with SNc dopamine neurons.
Cre dependent conditional expression of ChR2 was utilized in the creation of a floxed-STOP ChR2 mouse line (R26::ChR2-EGFP) (Katzel et al., 2011
), in which a mammalian codon-optimized ChR2(H134R)-EGFP driven by the CMV early enhancer/chicken β-actin (CAG) promoter and a floxed-STOP cassette was targeted to the Rosa26 locus by homologous recombination. After Cre-mediated excision of the floxed-STOP cassette, recombination brings the ChR2-EGFP coding sequence in frame with an initiating ATG in the loxP site, resulting in translation of ChR2-EGFP with an 11-amino-acid N-terminal ‘loxP tag’ (MYAIRSYELAT). After crossing to a Gad2-ires-CreERT2 mouse line, ChR2-EGFP was expressed in all main subclasses of cortical GABAergic interneurons, but undetectable in αCaMKII-positive pyramidal neurons, following tamoxifen induction. The relatively low level expression of ChR2 required the use of homozygous mice that were fed with retinol, as well as longer stimulating laser light pulses (20 ms, 2 mW) to evoke spiking than those used to activate virally transduced or in utero
transfected neurons. In these ChR2-positive cortical interneurons, under individual light pulses average peak photocurrents were ~190 pA, and average spike latency was ~16 ms. The light pulses were only able to elicit spiking in perisomatic areas, not in dendritic or axonal arborizations. Optical raster stimulation was used to map synaptic inputs from ChR2-positive interneurons to ChR2-negative pyramidal neurons in three cortical regions (M1, S1 and V1). It was found that the most common circuit motif is the lateral, intralaminar inhibition, supporting the view that inhibition is largely local, intralaminar and uniform across areas. However, rarer translaminar inhibitory inputs to subsets of layer 2/3 or layer 5 pyramidal neurons were also identified, predominantly in area V1.
The first functional Halo (NpHR) transgenic mice (orexin
/Halo) were generated using the 3.2 kb human prepro-orexin promoter by pronuclear injection (Tsunematsu et al., 2011
). Three founder lines showed sufficiently strong expression of Halo, and among these, line 5 showed the highest expression rate (94% of orexin neurons) and was used for the study. Interestingly, orexin neurons expressing Halo did not show blebbing or other features indicative of inappropriate trafficking. Maximum orange light illumination (586 nm, 4 mW) generated hyperpolarization of ~11 mV and photocurrents of ~6 pA in orexin neurons, and was able to inhibit either spontaneous or depolarizing current injection-induced action potential firing. Acute inhibition of orexin neurons in vivo
using orange LED light through optical fibers results in time-of-day-dependent induction of slow-wave sleep and in reduced firing rate of dorsal raphe neurons in an efferent projection site.
We have created four new mouse lines with high-level and Cre-dependent expression of ChR2(H134R) (Nagel et al., 2005
) fused to either tdTomato (Ai27; ChR2(H134R)-tdTomato) or EYFP (Ai32; ChR2(H134R)-EYFP), as well as a modified version of Arch (Ai35; Arch-EGFPER2) (Chow et al., 2010
) or eNpHR3.0 (Ai39; eNpHR3.0-EYFP) (Gradinaru et al., 2010
). These lines were generated using a transgenic expression strategy previously employed to generate a set of fluorescent reporter lines (Madisen et al., 2010
), in which the expression cassette, which includes (from left to right) the CAG promoter, the floxed-STOP cassette, the transgene, and a WPRE sequence, was targeted to the Rosa26 locus by homologous recombination. After crossing to several Cre lines, including Emx1
-Cre and Pvalb
-ires-Cre, all optogenetic reporter genes were found to be strongly expressed, by in situ
hybridization (ISH) and native fluorescence, in Cre-defined areas and cell types. Peak photocurrents reached nA range in both Ai27 and Ai32 ChR2 mice and 100–300 pA range in Ai35 Arch-ER2 and Ai39 eNpHR3.0 mice (unpublished data). The yet-to-published results demonstrate that robust, selective optical activation and silencing can be achieved in different neuronal cell types from different brain regions in all these mice, using a variety of photo-stimulation paradigms both in vitro
on brain slices and in vivo
in awake, behaving animals.
Transgenic expression of optical indicators
Genetically encoded calcium indicators (GECIs) offer unique opportunities to study calcium dynamics and cell signaling under a variety of physiologically relevant conditions. Since their inception, GECIs have undergone several rounds of optimization and are being increasingly used to monitor neuronal activities. In most applications, the GECIs have been produced in cells using viral approaches. Although several groups were successful in creating transgenic lines that expressed early-generation GECIs in non-neuronal tissues (Hara et al., 2004
; Ji et al., 2004
), the first mouse lines to express a functional GECI in the brain were two in which the indicators [inverse pericam (IP) and camgaroo-2 (Cg-2)] were transcribed from TRE-containing promoters following mating to the αCaMKII-tTA mouse line (Hasan et al., 2004
). Two different transgenic lines designed to express GCaMP2 in the brain were also generated and have been used to monitor the activities of different cell populations, including cerebellar granule cells (Diez-Garcia et al., 2007
; Diez-Garcia et al., 2005
), and cells in the olfactory system (Chaigneau et al., 2007
; He et al., 2008
). A troponin C-based sensor, CerTN-L15, was also expressed under the Thy1
promoter in transgenic mice, and exhibited Ca2+
responses in cortical pyramidal neurons both in vitro
and in vivo
(Heim et al., 2007
). Elucidation of the crystal structure of G-CaMP2 led to rationally designed changes in the molecule, resulting in GCaMP3, which demonstrates increased brightness, stability, and dynamic range (Tian et al., 2009
) as compared to G-CaMP2. No transgenic lines have yet been published that describe expression of GCaMP3 in any cell type. However, we’ve recently generated a Cre-dependent GCaMP3 reporter mouse that utilizes the same Rosa26
-based expression strategy we’ve successfully implemented for expressing other genetic tools. Preliminary data indicate that large calcium transients were observed with neuronal firing (Zariwala et al., unpublished data). Below we provide an overview of the published transgenic mouse lines expressing GECIs (see for a summary).
Transgenic mouse lines expressing genetically encoded calcium indicators
Among the first transgenics to express a GECI in the brain were two series of mice that carried randomly integrated cassettes for Tet-inducible expression of camgaroo-2 (Cg-2) or inverse pericam (IP) (Hasan et al., 2004
). Following mating to αCaMKII-tTA mice, many low-expressing lines exhibited punctate fluorescence inside cell bodies, while a few high-expressing lines showed more uniform cellular fluorescence. Functional Ca2+
responses in some of the highly-expressing lines were demonstrated in cells from several tissues. In hippocampal and cortical slices, short trains of stimuli evoked fluorescence changes (ΔF/F) in both Cg-2 and IP mice in the range of 2–8% by wide-field (WF) microscopy (with a CCD camera) and 10–100% by 2-photon (2P) microscopy. In retina light-evoked fluorescence changes were seen in whole-mount preps of IP-expressing mice (10% 2P), whereas no changes were detected in preps of Cg-2 mice. Finally, in vivo
imaging of the olfactory bulb showed odor-evoked fluorescence changes in both Cg-2 and IP-expressing mice (1–8% WF).
An improved version of the indicator yellow cameleon, YC3.60, created by using a circularly permuted YFP (cpYFP), exhibited a larger dynamic range and SNR in biochemical assays than previous versions of the molecule (Nagai et al., 2004
). A transgenic mouse line was made in which a modified CAG promoter (CAGGS) was used to drive expression of membrane-localized YC3.60. Upon tetanic stimulation of the Schaffer collateral/commissural pathway in these mice, a significant increase in FRET signal ([Ca2+
]) was evoked in area CA1 (ΔF/F of 2–3% by WF microscopy), and oscillatory [Ca2+
] in area DG (ΔF/F of 1–2% by WF microscopy). However, it was noted that the dynamic range of the indicator in cells of the CNS of these mice was greatly reduced from what had been observed in in vitro
The Kv3.1 potassium channel promoter was used to direct expression of G-CaMP2 in a defined subpopulation of neurons of the transgenic mouse line 846 (Diez-Garcia et al., 2005
). In the cerebellar cortex, G-CaMP2 was expressed exclusively in granule cells, where it reported presynaptic Ca2+
signals. In cerebellar slices, electrical stimulation in the molecular layer induced an increase in fluorescence in a beam-like area along the parallel fibers (ΔF/F of ~0.14% for a single stimulus, ~3% for 8 stimuli, and ~4% for 30 stimuli, WF microscopy). Stimulation at the granular layer induced both a local response and a beam-like response in the molecular layer. At high magnification (60×) in which brightest fluorescence was better localized, stimulations (10 pulses at 100 Hz) at either the molecular layer (antidromic activation) or the granular layer (orthodromic activation) both resulted in 5–25% ΔF/F in the target areas.
In subsequent studies on a subline of 846 (846HB) (Diez-Garcia et al., 2007
), direct stimulation (10 pulses at 100 Hz) of parallel fibers in the molecular layer of cerebellar slices evoked a ~50% ΔF/F through 2-photon laser-scanning microscopy, which was larger than what was obtained with 1-photon laser-scanning microscopy (~30% ΔF/F). Ca2+
signals were also detected in the cerebellar molecular layer in vivo
by both whole-field and 2-photon fluorescence imaging. Stimulations of parallel fibers in the molecular layer (10 pulses at 100 Hz) induced a ~3% whole-field fluorescence change that was clearly distinguishable from wild-type mice, as well as up to 50% ΔF/F by 2-photon imaging across responsive areas. Furthermore, using these transgenic mice, Ca2+
transients in the parallel fibers demonstrated presynatically-expressed long-term plasticity (both preLTP and preLTD) at the PF- Purkinje neuron synapses (Qiu and Knopfel, 2007
The Kv3.1-G-CaMP2 mouse line (846) has also been used in studies of the olfactory system, where G-CaMP2 was shown to be expressed in the mitral cells, tufted cells, and some juxtaglomerular cells in the olfactory bulb (Chaigneau et al., 2007
; Fletcher et al., 2009
). Two-photon imaging detected odor induced Ca2+
responses in the glomeruli that were odor specific, concentration dependent, and that could be blocked by superfusion of glutamate receptor antagonists. Glomeruli Ca2+
signals reflected activation of multiple mitral cells synchronized during population bursts, and stimulation of individual external tufted (ET) cells could drive population bursts of mitral cells within the same glomerulus (De Saint Jan et al., 2009
). These mice were employed to establish an odor-evoked sensory map with single glomerulus resolution, which reflected exclusively the activity of olfactory bulb neurons postsynaptic to sensory afferents (Fletcher et al., 2009
). In these G-CaMP2-based postsynaptic odor maps, different odorants activated distinct but overlapping sets of glomeruli. Increasing odor concentration increased both the response amplitude of individual glomeruli as well as the total number of activated glomeruli. Furthermore, the G-CaMP2 response displayed a fast time course that enabled analysis of the temporal dynamics of odor maps over consecutive sniff cycles.
In another transgenic mouse line that employed G-CaMP2, the indicator was expressed from the Tet-inducible promoter (He et al., 2008
). Following mating of this line with the OMP-ires-tTA line, G-CaMP2 expression was restricted to the olfactory sensory neurons in both the main olfactory epithelium and the vomeronasal organ (VNO). In VNO slices, diluted female or male urine samples evoked large Ca2+
transients (ΔF/F of 20% to >100%) in individual VNO neurons by 2-photon imaging. Diverse combinatorial activation patterns of VNO neurons were observed in response to gender, strain or individual specific pheromone stimuli.
Troponin C (TnC), the Ca2+
sensor protein in skeletal and cardiac muscle, was used as the basis to engineer a modified calcium sensor named CerTN-L15. Transgenic mice were generated to express CerTN-L15 under the Thy1
promoter (Heim et al., 2007
). In transgenic line C, which had the highest level expression of the transgene, the indicator was widely expressed in many types of neurons, most prominently in the pyramidal neurons of the hippocampus and the neocortex. In cortical slices through 2-photon imaging, although fluorescence changes caused by single action potentials were not reliably detected, brief trains of multiple action potentials evoked clear changes in the ratio of Citrine/Cerulean fluorescence (ΔR/R), with a linear relationship between the number of action potentials and the ΔR/R (extrapolated ΔR/R = ~4% per action potential). Iontophoretic glutamate applications in layer 2/3 neurons of the visual cortex in vivo
evoked cellular Ca2+
signals that were similar to those evoked in slices, as well as dendritic Ca2+
transients with ~49% ΔR/R. The TnC-based sensors may be advantageous compared to those based on calmodulin (CaM), in that CaM interacts extensively with other intracellular neuronal proteins, which could make the in vivo
functionality of CaM-based Ca2+