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Cell-type-specific expression of optogenetic molecules allows temporally precise manipulation of targeted neuronal activity. Here we present a toolbox of 4 knock-in mouse lines engineered for strong, Cre-dependent expression of channelrhodopsins ChR2-tdTomato and ChR2-EYFP, halorhodopsin eNpHR3.0, and archaerhodopsin Arch-ER2. All 4 transgenes mediate Cre-dependent, robust activation or silencing of cortical pyramidal neurons in vitro and in vivo upon light stimulation, with ChR2-EYFP and Arch-ER2 demonstrating light sensitivity approaching that of in utero or virally transduced neurons. We further show specific photoactivation of parvalbumin-positive interneurons in behaving ChR2-EYFP reporter mice. The robust, consistent, and inducible nature of our ChR2 mice represents a significant advancement over previous lines, whereas the Arch-ER2 and eNpHR3.0 mice are the first demonstration of successful conditional transgenic optogenetic silencing. When combined with the hundreds of available Cre-driver lines, this optimized toolbox of reporter mice will enable widespread investigations of neural circuit function with unprecedented reliability and accuracy.
A major challenge in neuroscience is to understand how brain functions are mediated by particular cell types within neural networks. Dissection of such complex networks requires the ability to manipulate the activities of specific cell types and to examine the resulting effects. One of the most exciting recent innovations in experimental neuroscience has been the development of light-activated channels or pumps, derived from microbial photosynthetic systems, to modulate neural activity, known as optogenetics. The best-known prototypes for the application of optical control in neurons include the neural-activating cation channel, channelrhodopsin-2 (ChR2) 1-3, and the neural-silencing chloride transporter halorhodopsin (NpHR) 4, 5 and proton pump archaerhodopsin-3 (Arch) 6. These and related optogenetic molecules 7 allow activation or silencing of neurons with unprecedented specificity and excellent temporal precision on a millisecond scale, and are in widespread use.
In mice, cell-type-specific genetic manipulation is most widely achieved through the Cre/lox recombinase system. Hundreds of Cre mouse lines, generated in individual labs and through large-scale efforts 8-10, have been established to direct specific gene expression or deletion in a wide range of cell types or populations throughout the nervous system. A major challenge to developing optogenetic tools is the need to express high levels of the opsins, due to the relatively small optical current mediated by each opsin molecule. For this reason, opsin genes have been most often introduced in vivo using recombinant viral vectors or by in utero electroporation (IUE). By delivering an adeno-associated virus (AAV) that expresses an opsin in a Cre-dependent manner, e.g. using floxed-stop or floxed-inverse (FLEX) cassettes 11, 12, it is possible to virally deliver an opsin to a brain region, where only Cre-positive cells will activate expression of the opsin. Although successful for many applications, these approaches possess intrinsic limitations. They can result in incomplete coverage of neurons within the region, which may limit experiments requiring complete labeling (e.g. neural silencing), and variable opsin expression levels across cells from the injection center out. The variability in the number and location of opsin-expressing cells between animals necessitates laborious validation for each animal, introducing variability in data interpretation. The brain targets of interest may be very small, very large, or hard for the virus to access for other reasons (e.g. a difficult location for injection, a particular cell type that cannot be infected by any serotype of virus, or during brain development).
A transgenic mouse approach can overcome many of these limitations. However, exogenous opsin gene expression in transgenics is typically regulated by a specific linked promoter, and so, is predetermined to occur in a particular and fixed cell population 13-19. A Cre-dependent system of opsin expression would exploit the abundant resource available Cre-driver lines constitute and would offer a powerful approach for controlling the activity of a wide range of cells. However, transgenic mice with robust Cre-dependent expression of opsins have been difficult to generate, as indicated by recent characterization of a ChR2 Cre-reporter 20. To date, no transgenic line with Cre-dependent expression of a silencing opsin has been described. Efforts to overcome the limitations of early versions of silencing opsins, such as protein aggregation and low conductance 21, has led to the development of newer optical silencing molecules, including eNpHR 22, eNpHR3.0 23, and various forms of Arch 6, 24, with great improvement in membrane expression and related increase in photoconductance, making reliable genetic silencing achievable now.
Here we report the creation of a toolbox of 4 new mouse lines with high-level and Cre-dependent expression of ChR2(H134R)-tdTomato, ChR2(H134R)-EYFP, Arch-EGFP-ER2, or eNpHR3.0-EYFP. Inducible expression of these opsins is driven by a unique expression cassette in a modified Rosa26 locus, which we recently showed capable of mediating efficient fluorescent labeling 9. Here we demonstrate for the first time that high-performance optogenetic manipulation in Cre-dependent transgenic mice is enabled for all the classes of opsin molecules using this optimized strategy.
For all 4 lines, we found that cortical pyramidal neurons were highly responsive to either light activation (ChR2s) or light inhibition (eNpHR3.0, Arch-ER2), in both in vitro brain slice preparations and in vivo brains of awake animals. In addition, we found that light activation of hippocampal and reticular parvalbumin (Pvalb)-positive interneurons, as well as cortical rhythms resulting from synchronized reticular Pvalb+ neuron activation, can be readily achieved in behaving ChR2-EYFP mice. These results demonstrate that selective optical activation and silencing can be applied to different cell types in different brain regions in these mice, using a variety of photostimulation paradigms. Thus, when combined with the plethora of publically available Cre-driver lines, these transgenics should greatly facilitate the study of various neuronal cell types including those inaccessible to previous approaches.
We previously demonstrated strong, ubiquitous, Cre-dependent expression of fluorescent markers from a modified Rosa26 locus by incorporating a CAG promoter and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) 9. Using the same expression strategy, we created 4 new Cre-dependent mouse lines that express optogentic tools: the H134R mutant version of ChR2 25 fused to either tdTomato (Ai27; ChR2(H134R)-tdTomato) or EYFP (Ai32; ChR2(H134R)-EYFP); a modified version of Arch (Ai35; ss-Arch-EGFP-ER2, abbreviated as Arch-ER2) 6; or eNpHR3.0 (Ai39; eNpHR3.0-EYFP) 23 (Fig. 1a). ChR2(H134R) was chosen because it produces larger conductance changes compared to wild-type ChR2, likely due to its slower deactivation kinetics. Both Arch-ER2 and eNpHR3.0 have Kir2.1 ER-exporting signals which were shown to enhance proper expression of microbial opsin proteins to the cell membrane 6, 21-23.
To examine Cre-dependent expression of the optogenetic reporters, each line was bred with Emx1-Cre. Consistent with Emx1-Cre's recombination pattern, all mice displayed strong native fluorescence throughout cortex and hippocampus (Fig. 1b). In each line, the fluorescent fusion proteins were primarily localized to the cell membrane with minimal accumulation in the cytoplasm, which was best seen in the cell body layer of all hippocampal subfields (CA1, CA3, and dentate gyrus) where the fluorescence was lower than in the dendritic layers and was ring-shaped (Fig. 1b-c). Axon fibers extending from the cortex and hippocampus, as well as their termination zones (e.g. thalamus), were also strongly labeled (Fig. 1b). Axon fiber fluorescence in Emx1-Cre;Ai39 mice appeared weaker than in other lines, indicative of possible lower protein expression. The mRNA expression of the opsin-fusion genes was mapped by in situ hybridization (ISH) on brain sections (Fig. 1d). All showed strong ISH signals in the cortex and hippocampus at the single-cell level, similar to expression seen in our Cre reporter mouse lines that express fluorescent proteins 9.
In the absence of Cre, no leaky expression at mRNA or fluorescence level was seen in Ai27 and Ai32 mice, although some leakage at mRNA level was seen in Ai35 and Ai39 mice (Supplementary Figs. 1a and 2a). In addition to Emx1-Cre, strong Cre-dependent expression of opsin-fusion genes was also seen throughout the brain in other Cre-driver crosses (e.g. Pvalb-IRES-Cre, Camk2a-CreERT2, Chat-IRES-Cre) (Supplementary Figs. 1b, 2b and 3), consistent with previous studies of our Cre-dependent fluorescent reporter mice 9. Plasma membrane targeting of all 4 transgenes was also seen in all cases with no detectable intracellular protein aggregates, as shown in the Pvalb+ interneurons in the cortex of the 4 types of Pvalb-IRES-Cre/reporter mice (Supplementary Fig. 1b). Long-term expression of these optogenetic transgenes in the various Cre-defined cell populations did not produce observable toxicity (Supplementary Fig. 4). The robust and widespread opsin-fusion expression observed in these multiple lines thus suggests that a variety of cell populations will be amenable to photo-manipulation by these Cre-dependent optogenetic tools.
We investigated the photoexcitability of cortical pyramidal neurons from Emx1-Cre;Ai27, Emx1-Cre;Ai32 mice (abbreviated as E-Ai27 and E-Ai32), and Ai32 alone mice not crossed to any Cre, by whole-cell recordings in the barrel cortex of acute brain slices with a previously established photostimulation paradigm 26, 27 (Fig. 2a), and compared them to neurons in which ChR2 was expressed by in utero electroporation (IUE). ChR2-expressing neurons showed normal resting membrane potentials (E-Ai27, -65.7 ± 1.4 mV, n = 14; E-Ai32, -65.4 ± 1.4 mV, n = 9; Ai32 (-Cre), -66.6 ± 2.2 mV, n = 9; IUE, -66.0 ± 2.4 mV, n = 6; mean ± s.e.m.). For each trial, a series of 1-ms pulses of constant-power blue light (473 nm) was applied through an air objective, sequentially on an 8 × 16 grid pattern overlaying cortical layers 1-5 (Fig. 2a). The grid covered the soma, dendrites, and parts of the axonal arbor of the recorded layer 2/3 pyramidal neuron (Fig. 2b). In voltage clamp mode, large photocurrents were evoked in E-Ai27 and E-Ai32 cells with modest laser powers (Fig. 2b). With high laser powers (1350-1700 μW), the large photocurrents (peak current for Ai27: 1.51 ± 0.24 nA, n = 10; Ai32: 2.01 ± 0.04 nA, n = 7) were often accompanied by spikes. Even under high power stimulation conditions, no detectable photocurrent was recorded from Ai32 (-Cre) control cells (Fig. 2b, n = 9). In current clamp mode, action potentials (APs) were evoked in 12 of 14 E-Ai27 cells (2 cells showed subthreshold or no response), in 9 of 9 E-Ai32 cells which is comparable to IUE cells 26, 28, and in 0 of 9 Ai32 (-Cre) cells (Fig. 2c-d).
To compare the photosensitivity, each cell was stimulated with a series of varying laser powers to determine the threshold for evoking an AP (Fig. 2e). We found that E-Ai27 cells showed substantial heterogeneity in their thresholds for spiking, whereas E-Ai32 cells demonstrated consistently low thresholds (Fig. 2e-f). The average threshold powers were: E-Ai27, 919 ± 146 μW (ranging 550-1720 μW, n = 12); E-Ai32, 67 ± 6 μW (ranging 55-116 μW, n = 9); IUE, 56 ± 17 μW (ranging 8-128 μW, n = 7) (Fig. 2e). We further found that high power stimulation consistently evoked APs on more sites of the grid for E-Ai32 cells than E-Ai27 cells (Fig. 2g) and with shorter spike latencies (Fig. 2h). Notably, low laser powers evoked APs in E-Ai32 cells similar to IUE cells, but E-Ai32 cells showed less variability in the number of sites that could trigger an AP (Fig. 2i).
Photoexcitation of axons is desirable for stimulating long-range postsynaptic neurons. The subcellular distribution of ChR2 will also influence the spatial resolution of photostimulation in brain slices and in vivo 29. Antidromic APs triggered in axons can be distinguished from those triggered in the somata and dendrites, because somatic/dendritic APs are associated with a slow charging phase preceding the AP threshold whereas axon-initiated APs arrive in the soma without prior charging 30 (Fig. 2c). By analyzing the charging phase, each AP was categorized as originating from either somatic/dendritic or axonal stimulation (Fig. 2d). Under our photostimulation conditions both E-Ai27 and E-Ai32 cells were preferentially excited in axons, whereas E-Ai32 cells, similar to IUE cells, were also more readily excited in the somata and dendrites than E-Ai27 (Supplementary Fig. 5). In sum, our results demonstrate that whereas E-Ai27 cells are less sensitive to photostimulation, the light response properties of E-Ai32 cells are comparable in many aspects to those of IUE ChR2-expressing cells.
To test light-evoked neuronal silencing, we performed whole-cell recordings from layer 2/3 or layer 5 cortical pyramidal neurons (Fig. 3a) in acute slice preparations taken from adult Emx1-Cre;Ai35 or Emx1-Cre;Ai39 mice (abbreviated as E-Ai35 or E-Ai39). In all pyramidal neurons tested (29/29), illumination with a 200-μm optical fiber coupled to a 593-nm yellow laser caused marked hyperpolarization from the resting membrane potential. The response was very similar to a hyperpolarizing current injection prior to the light pulse (Fig. 3b). Recorded neurons exhibited a variety of responses to hyperpolarizing currents injected under current clamp conditions. Some exhibited Ih currents and/or rebound firing of APs in response to a transient application of a hyperpolarizing current, while others did not. In each case, the effect of the input current could be effectively reproduced by light (Fig. 3b). Upon repeated cycles of light stimulation (23 mW mm-2), the extent of hyperpolarization diminished to about two-thirds of the initial value, then reached a plateau in E-Ai39 cells (Fig. 3c). The E-Ai39 cells could recover from such diminished response to >90% original level after ~5-min rest between the cycles. Unlike E-Ai39, the E-Ai35 neurons did not exhibit diminishing effects with repeated light stimulation (Fig. 3c).
Both E-Ai35 and E-Ai39 cells showed larger hyperpolarization with increased light intensities (Fig. 3d). The mean maximal hyperpolarization (measured at 23 mW mm-2) was 26.39 ± 2.67 mV for E-Ai35 (n = 15) and 12.96 ± 1.39 mV for E-Ai39 (n = 14). Under voltage clamp conditions, light-induced currents were corresponding to light intensity and were highly reproducible from cell to cell (Fig. 3e-f). The photocurrents attained in E-Ai35 neurons at low and high light levels were 2-3 fold greater than those observed in E-Ai39 neurons (1.7 mW mm-2: p < 0.05, n = 5 for E-Ai35, n = 7 for E-Ai39; 23 mW mm-2: p < 0.01, n = 8 for E-Ai35, n = 9 for E-Ai39) (Fig. 3f). APs induced under current clamp by constant positive current injections were rapidly and reversibly inhibited by light illumination (23 mW mm-2) in all trials in both E-Ai35 and E-Ai39 neurons (Fig. 3g). We conclude that Ai35-expressed Arch-ER2 is somewhat more efficient at generating photocurrents than Ai39-expressed eNpHR3.0, but that both transgenically-expressed opsins are effective tools for the silencing of neural activity.
To ensure that no physiologically significant opsins are expressed from the un-induced reporter loci, we examined light-induced responses in cortical pyramidal neurons (n = 4) of Ai35 mice, as well as cortical (n = 4) and hippocampal CA1 (n = 3) pyramidal neurons of Ai39 mice, in the absence of any Cre allele. No light-induced changes were observed in the Ai35 (-Cre) cells at all, either at resting membrane potential (Fig. 3h-i) or when activated by a positive current injection (Fig. 3j), even with the highest intensity (23 mW mm-2). Similarly for Ai39 (-Cre) cells, light (593 nm, 23 mW mm-2; or 640 nm, 24 mW mm-2) caused no or negligible change in membrane potential (-0.02 ± 0.08 mV, n = 7, Fig. 3i). This is consistent with the leaky mRNA but no leaky protein expression in Ai39 (-Cre) mice (Supplementary Figs. 1a and 2).
In addition to the yellow light (593 nm) which is near the excitation maximum of the Arch and NpHR proteins 4, 6, 31, we also tested neural silencing by two alternative light sources, a 640-nm red laser and a white LED, that offer several practical advantages. Red lasers are less expensive and more easily modulated than the yellow lasers. Though somewhat red-shifted relative to the excitation peaks of Arch and NpHR, longer wavelengths are also less prone to attenuation by light scattering, so the off-peak excitation may be partly offset by improved tissue penetration. Red light also offers better spectral separation from the blue light used to activate ChR2, so they may be better suited for binary control of activity 4, 32. LEDs are inexpensive light sources and are potentially portable or implantable for experiments in moving animals.
These alternative light sources efficiently silenced both E-Ai35 and E-Ai39 cortical pyramidal neurons (Fig. 4). Both the LED and 640-nm red laser produced an intensity-dependent drop in membrane potential (Fig. 4b,e,g,i), and effectively silenced current-induced spiking (Fig. 4c,f,h,j). At similar powers, the 640-nm light response of E-Ai35 neurons was ~43% of that obtained with 593-nm light (Figs. 3d and and4e),4e), while the 640-nm light response of E-Ai39 neurons was ~100% of that obtained with 593-nm light (Figs. 3d and and4i),4i), consistent with the red-shifted spectrum reported for eNpHR3.0 23.
In order to test the effectiveness of Emx1-Cre;Ai35 mice as a model for inhibiting neural network activity, we tested light-mediated silencing of a well-described monosynaptic pathway from area CA3 to area CA1 of the hippocampus (Fig. 5a) 33. In this model, firing of hippocampal neurons was induced in brain slice preparations by elevated K+, and population activity was recorded from CA1 (Fig. 5b). Focused illumination of the CA3 region produced an immediate drop in CA1 activity (Fig. 5c). With sustained illumination, CA3 firing increased, but remained below baseline level. Partial release from silencing may occur over time because of compensatory effects in the network and/or because only a portion of the CA3 neurons with outputs to CA1 are inhibited by the focal illumination. Following light off, firing rates returned quickly to baseline, and there was no rebound of spiking activity.
Dual recording from CA1 and CA3, combined with focal illumination of CA1, confirmed that these results were consistent with the silencing of a unidirectional synaptic input from CA3 to CA1 (Fig. 5d-e). First, illumination of CA1 had no effect on population activity in CA3. By contrast, direct illumination of CA1 caused immediate and nearly complete silencing of this region, and a large rebound increase in firing was observed upon recovery from direct illumination of CA1. In addition, the extent of CA1 inhibition was dependent on the area of illumination on CA3, under conditions of constant light intensity per unit area (Supplementary Fig. 6). We conclude that illumination of CA3 results in the selective inhibition of synaptic inputs to CA1, thus demonstrating the potential of Ai35 (and Ai39) mice for optogenetic studies of brain circuitry.
To evaluate the effectiveness of light modulation in vivo in these transgenic mice lines, we performed extracellular recordings in awake, head-fixed adult mice. We estimated that light intensity at the electrode recording site to be 1-5% of that at the optical fiber tip, which was 500-900 μm above the recording site, based on the Monte Carlo simulation of light attenuation in brain tissues 6.
In Emx1-Cre;Ai32 mice, we recorded a total of 16 units, including 7 single units and 9 multi units. Of the 7 single units, firing rates significantly increased in 6 units (Fig. 6a), and decreased in 1 unit, upon blue light (473 nm) illumination (0.8 mW out of a 100-μm optical fiber, corresponding to ~100 mW mm-2 at the fiber tip and ~2 mW mm-2 at the recording sites). The light induced spiking is fast, reliable and precise after each pulse illumination (Supplementary Fig. 7). Precise activation was observed even when the fiber tip was 1.5-2 mm away from the recording site, where light intensity is expected to drop to ~0.17% of that at the tip. Of the 9 multi units, firing rates significantly increased in 8 units, and decreased in 1 unit. Light illumination and recording was also performed in an Ai32 (-Cre) mouse as a negative control. Of the 3 cortical neurons recorded, none were modulated by light at the highest power. In Emx1-Cre;Ai27 mice, we recorded a total of 11 units, including 4 single units and 7 multi units. Of the 4 single units, firing rates significantly increased in only 1 unit, and decreased in the other 3, with the same blue light illumination (2.2 mW, ~280 mW mm-2 at the 100-μm fiber tip, and ~5 mW mm-2 at the recording sites). However, all 7 multi units increased their firing rates. Overall, the single and multi units with increased firing rates showed an enhancement of 350-1400% from the baseline for E-Ai32 (768 ± 94%, n = 14), and 150-350% for E-Ai27 (198 ± 25%, n = 8) (Fig. 6b). The magnitude of light-induced activation in E-Ai32 is significantly higher than that in E-Ai27, consistent with that observed in vitro (Fig. 2).
To evaluate the efficiency of optical silencing in vivo mediated by eNpHR3.0 and Arch-ER2, we used Emx1-Cre;Ai35, Camk2a-CreERT2;Ai35, and Camk2a-CreERT2;Ai39 mice. Following tamoxifen induction, Camk2a-CreERT2 drives eNpHR3.0 or Arch-ER2 expression in majority of cortical pyramidal neurons similar to Emx1-Cre (Supplementary Fig. 1) 9. In an Emx1-Cre;Ai35 mouse, we recorded a total of 10 single units in cell attached mode with glass electrodes. All single units significantly reduced their firing rates upon green light illumination (532 nm, 16 mW, ~470 mW mm-2 at the 200-μm fiber tip, and ~13 mW mm-2 at the recording sites) (Fig. 6c). The recorded neurons showed a reduction of 83-100% from the baseline firing rates (93 ± 2%, n = 10) (Fig. 6e, left panel). Light-induced silencing was instantaneous, with 0 ms latency for 9 of the 10 neurons recorded. The population latency was 1 ± 1 ms from light onset (Fig. 6e, right panel). Similar results of complete silencing of single unit activities were observed in a tamoxifen-induced Camk2a-ERT2;Ai35 mouse (93 ± 7% silencing at ~10 mW mm-2 at the recording sites, n = 2). Light illumination and recording was also performed in an Ai35 (-Cre) mouse as a negative control. Of the 8 cortical neurons recorded, none were modulated by light at the highest power used (~16 mW mm-2 at the recording sites).
In a tamoxifen-induced Camk2a-CreERT2;Ai39 mouse, we recorded a total of 13 single units. Of these, 7 significantly reduced their firing rates (Fig. 6d), 3 did not change, and 3 increased their firing rates upon green light illumination (11 mW, ~1400 mW mm-2 at the 100-μm fiber tip, and ~30 mW mm-2 at the recording sites). The single units with reduced firing rates showed a reduction of 83-97% from the baseline (90 ± 2%, n = 7), with 0-20 ms latency from light onset (8.6 ± 4.0 ms) (Fig. 6e). The magnitude of silencing in Ai39 mice with 30 mW mm-2 light is comparable to that observed in Ai35 mice with 10 mW mm-2 light (p = 0.4), but the latency of silencing in Ai39 mice is significantly longer than that in Ai35 mice (p < 0.05). The observed incomplete inhibition could result from the neurons that did not express Camk2a-CreERT2, and thus were disinhibited or activated through neural network mechanisms, consistent with previous observations using viral labeling method in vivo 24.
We then compared the responses of the same single units to different light intensities. These single units in both Emx1-Cre;Ai35 and Camk2a-CreERT2;Ai39 mice showed less inhibition at lower light intensities (Fig. 6f). In Ai35, firing rates were reduced by 49 ± 10% at 5 mW mm-2, and by 93 ± 2% at 13 mW mm-2 (n = 10, p = 0.001). In Ai39, firing rates were reduced by 62 ± 9% at 10 mW mm-2, and by 90 ± 2% at 30 mW mm-2 (n = 7, p = 0.02).
To examine the photoexcitability in an inhibitory neuronal type, we performed extracellular recordings in awake behaving Pvalb-IRES-Cre;Ai32 mice using a custom-designed optoelectronic probe 34 containing 5-20 μm optical fibers that deliver locally focused light (~1 mW at the tip). For an independent assessment of the specificity of Pvalb+ neuron activation in these mice, two brain areas, the hippocampal CA1 region and the thalamus, were chosen because in these areas interneurons and principal cells can be reliably separated by physiological means 35-37. In the hippocampus, the neuron with the typical autocorrelogram (ACG) of fast firing perisomatic interneurons 38 was directly activated by both single pulses and the sinus pattern (Fig. 7a). In contrast, the bursting, putative pyramidal cell was silenced at the time of the firing epoch of the activated neuron. Using previously established waveform criteria, trough-to-peak time and the width of the spike of the wide-band (1 Hz – 5 kHz) unit at 20% of its peak amplitude 35, 39, we were able to segregate all recorded neurons into two major groups (Fig. 7b and Supplementary Fig. 8). All of the light activated neurons fell into the 'narrow-spike category'. In another comparison, neurons were identified as excitatory or inhibitory neurons on the basis of putative monosynaptic excitation and inhibition estimated by their short-time cross-correlograms 35, 39, 40. Again, the two groups were segregated and all activated neurons fell into the physiologically identified interneuron cluster (Fig. 7b and Supplementary Fig. 8). These results show that ChR2-mediated activation was absent in CA1 pyramidal cells, and present in fast firing interneurons, corresponding to the perisomatic Pvalb+ interneurons.
In the thalamus, the characteristic low-threshold spike burst patterns of thalamocortical neurons can be reliably separated from the fast firing inhibitory interneurons of the reticular nucleus 37. In the mouse reticular nucleus the majority of interneurons are PVALB-immunoreactive 41. Simultaneous recordings from reticular and the adjacent thalamocortical neurons and their local optogenetic activation generated two separate groups. Neurons that were activated by either single pulses or sinusoid pattern had ACGs characteristic of reticular neurons, whereas bursty neurons were suppressed by light stimulation (Fig. 7c and Supplementary Fig. 9). Light-activated and light-suppressed neurons had significantly different burst index magnitudes (Fig. 7d). Thus, these findings establish further support for the specificity of ChR2-mediated activation in reticular Pvalb+ interneurons. Although these physiological methods cannot distinguish between Pvalb+ and other classes of interneurons 42, the physiological results are consistent with the previously demonstrated recombination specificity of Pvalb-IRES-Cre to Pvalb+ interneurons in both hippocampus and thalamus 9.
Since reticular inhibitory neurons are critical for pacing thalamocortical rhythm 37, we examined their light activation on neocortical activity of waking mice. Single pulse stimulation (5 ms, 20 mW) evoked a primary response, likely through rebound spike activation of thalamocortical neurons 36, 37, followed by several cycles of activity typical of thalamocortical reverberation (Fig. 7e, left panel). Repetitive activation of reticular neurons at 10 Hz effectively entrained the thalamocortical circuit 36 (Fig. 7e, right panel). These findings demonstrate that reticular neurons can be recruited effectively by local light stimulation in Pvalb-IRES-Cre;Ai32 mice to induce physiologically relevant cortical patterns.
We have established 4 new transgenic mouse lines with robust Cre-dependent expression of ChR2, Arch-ER2 or eNpHR3.0. Compared to a previously published R26ChR2-EGFP Cre-reporter mouse line 20, our Ai32 ChR2-EYFP-expressing line exhibits significantly greater light sensitivity. In the R26ChR2-EGFP mice, using very similar stimulation paradigms in cortical slices, very long and strong laser pulses (~20 ms, ~2 mW) were needed to induce single spiking in interneurons, and even so spiking could not be induced in pyramidal neurons. The necessity to use mice with homozygous floxed-ChR2 allele and to feed the mice with retinol was also reported. In contrast, the Ai32 mice possess excitability with 1-ms light pulses at intensities as low as 30 μW, much larger photocurrents, and much shorter latency to spiking, in pyramidal neurons. We further show that pyramidal neurons and interneurons are also readily excitable in vivo with low light in heterozygous animals. These greatly improved properties are likely to be proven critical in future in vitro and in vivo studies. Using such an efficient transgenic expression strategy is particularly essential for expressing the silencing opsins (Arch and eNpHR) to functional levels.
The transgenic targeting strategy used in the R26ChR2-EGFP line 20 is similar to our strategy described here. Both targeted the Rosa26 locus and used a strong and ubiquitous CAG promoter. But, two main differences may be responsible for the improved expression level in the new lines reported here. First, our expression cassettes included a WPRE sequence, which we previously showed to enhance protein expression of Rosa26-targeted transgenes 9. Second, while the R26ChR2-EGFP line incorporated a commonly-utilized floxed-stop cassette that contains the bacterial neomycin-resistant (neo) gene in front of the stop, we intentionally excluded the neo gene from our own floxed-stop cassette and instead placed it separately downstream from the transgene expression cassette. Although it is unclear whether the relocation of the neo gene is helpful to the significantly improved expression in our mice, it is well-known that integration of bacterial sequences into mammalian genomes could cause epigenetic modifications that affect expression of nearby genes.
Each of our optogenetic lines exhibited distinct photo-response properties that could be useful for different applications. Neurons from Ai32 appeared significantly more photosensitive than those from Ai27, both in vitro and in vivo. Although the basis for this difference between Ai27 and Ai32 neurons is unresolved, and no apparent difference in expression level or membrane localization was seen, we speculate that the bulkier tdTomato fluorescent tag in the Ai27 transgene may interfere with some channel properties. Although Ai32's photosensitivity seems desirable for most experiments, Ai27's red label and/or preferential axonal excitation may be advantageous in some applications 28, 30. Our in vitro and in vivo data on Ai35 and Ai39 neurons suggest that Arch-ER2 produces greater photocurrents and larger hyperpolarization than eNpHR3.0 under both green and yellow light. This could be due to the observed lower protein level for eNpHR3.0 even though the mice were generated using identical designs. However, due to its broad and red-shifted activation spectrum, eNpHR3.0 is equally effective as Arch-ER2 under red light, and may be preferable for dual-channel work together with blue light-responsive depolarizing opsins.
The kinetics of the in vivo light response of ChR2-expressing neurons in Ai27 and Ai32 mice were identical to those observed with virally expressed opsins, with a rapid increase in spiking at light onset, followed by a stable, lower steady-state firing level during light stimulation and often a period of suppression immediately following light termination 32. Photoinhibition of Arch-ER2 or eNpHR3.0-expressing neurons in Ai35 or Ai39 mice in vivo showed a near instantaneous reduction in firing rate, consistent with that observed with viral infection method 6. Many of the neurons recorded in Ai35 and Ai39 mice were nearly completely silenced. The homogenous and complete silencing in these mice presents a major advantage of the consistent expression achieved in these transgenic mice compared to viral infection method 6, 24.
An important question is the specificity of neuronal activity manipulation in the targeted populations. While this can be addressed by exhaustive anatomical multiple labeling methods, physiological verification of specificity is also useful. We have chosen two brain areas, the CA1 pyramidal layer and the thalamus because identification of principal cells and interneurons in these regions is possible by physiological means 35-40. While these methods cannot distinguish among the large family of interneurons 38, light-activated neurons displayed well-known features of short-duration spikes and high firing rates, typical of perisomatic Pvalb+ interneurons. Conversely, putative CA1 pyramidal neurons and thalamocortical cells with bursting properties and excitatory connections were never directly activated by light. Instead, the majority of them were suppressed by light stimulation. These excellent physiological-optogentic correlations in the intact brain support the cell type specificity of ChR2 activation. They also suggest that optogenetic activation of genetically labeled cell types, especially those difficult to distinguish by physiological or other means, will enable more refined in vivo identification and characterization of their functional properties 43.
Systematic expression characterization data from our previously generated fluorescent reporter lines (e.g. Ai14), crossed to dozens of different Cre-driver lines, have shown that Cre-dependent activation of transgene expression can be obtained in nearly all neuronal types 9. Thus, although data presented here are confined to the light responses of cortical/hippocampal pyramidal neurons and hippocampal/thalamic Pvalb+ interneurons, we believe it highly likely that these optogenetic tools will effectively modulate activity in a wide range of neurons. It should be noted that different types of neurons may have different excitability properties for both intrinsic and/or local circuitry reasons, and hence may require individual optimization of the conditions for activation and silencing.
Here we not only provide a novel set of transgenic tools with superior properties for both stimulating and silencing neuronal activity, we also demonstrate a transgenic expression strategy having several significant conceptual advantages. First, since the opsins are expressed as single copies in an identical manner from a consistent genomic environment, reliable comparisons of in vivo performance can be made between different opsin genes and different transgenic lines. Second, the Cre-dependent on-off switch for transgene expression effectively prevents or minimizes leaky expression in non-targeted cells while enables strong expression from the well-characterized CAG promoter in targeted cells, a clear advantage over the specific promoter-driven single transgenic approach in which the promoters used could have variable (sometimes unknown or uncharacterized) expression in both targeted and non-targeted cells. Third, because of the efforts we and others have taken to systematically characterize expression in Cre-driver lines 8, 9, especially when similar reporter lines are used, documented and publicly available information about Cre-recombination patterns (e.g. http://connectivity.brain-map.org/transgenic/search/basic) can advise researchers about the cell-type specificity of expression expected when using a particular Cre driver with these reporter lines. This is of great importance also considering that unintended ectopic Cre expression may exist to varied degrees in some Cre lines, and informed choice about appropriate Cre (or inducible Cre) lines for specific cell types need to be made. Finally, this proven expression system will also facilitate the rapid incorporation of newly engineered optogenetic variants, and once validated, apply them to all the Cre lines. This presents a "one-for-all" opportunity than expressing one opsin in one cell type at a time, and will further increase the range of optogenetic capabilities for investigating neural circuits and brain function.
All experimental procedures related to the use of mice were approved by the Institutional Animal Care and Use Committees of the Allen Institute for Brain Science, the Howard Hughes Medical Institute, Boston University, Seattle Children's Research Institute, or Rutgers University, in accordance with NIH guidelines.
Targeting constructs were generated using a combined gene synthesis (GenScript) and molecular cloning approach. ChR2(H134R)-tdTomato was synthesized based on the hChR2 sequence 26, and ChR2(H134R)-EYFP was synthesized based on the ChR2 sequence 3. Both fragments, as well as the ss-Arch-EGFP-ER2 6 and the eNpHR3.0-EYFP 23 fragments, were each cloned into a Rosa26-pCAG-LSL-WPRE-bGHpA targeting vector 9, in between LSL and WPRE sequences. LSL sequence contains specifically LoxP – Stop codons – 3x SV40 polyA – LoxP.
The targeting vectors were linearized and transfected into the 129/B6 F1 hybrid ES cell line G4 44. G418-resistant ES clones were first screened by PCR using primers spanning the 1.1 kb 5' genomic arm (forward primer: 5'-gggctccggctcctcagaga-3', reverse primer: 5'-atgccaggcgggccatttac-3'), and then confirmed by Southern blot analysis of HindIII digested DNA, which was probed with a 1.1 kb genomic fragment from immediately upstream of the 5' arm. Positive ES clones were injected into C57BL/6J blastocysts to obtain chimeric mice following standard procedures. Chimeric mice were bred with C57BL/6J mice to obtain germline transmitted F1 mice. The reporter mice can be bred with the Rosa26-PhiC31 mice (JAX Stock # 007743) 45 to delete the PGK-neo cassette in the germline of the mice.
Reporter mice were crossed to various Cre lines, including Emx1-Cre (JAX Stock # 005628), Camk2a-CreERT2 (JAX # 012362), Pvalb-IRES-Cre (JAX # 008069) and Chat-IRES-Cre (JAX # 006410). Expression of the reporter genes was assessed by both native fluorescence (without antibody staining) on perfused, microtomed sections and by ISH.
For ISH, the Allen Institute-established pipelines for tissue processing, probe hybridization, image acquisition, and data processing were used. The procedures were previously described 9, 46 and can be found at the Transgenic Mouse database (http://help.brain-map.org/display/mouseconnectivity/Documentation). Expression levels were analyzed with 2-tailed, paired or unpaired Student's t-test at an alpha level of 0.05.
P30 – P60 mice were used. Recordings on 300-μm thick barrel cortical slices were performed in the presence of 5 μM (R)-CPP (Tocris) and 10 μM NBQX (Tocris). Electrophysiology and stimulus conditions were as described 26. A series of 473-nm laser pulses (Crystal Laser) of 1-ms duration and 30-1770 μW (at specimen) for photostimulation was delivered through an air objective (4x; 0.16 NA; UPlanApo, Olympus) with a beam diameter of 6-20 μm (scattering in the tissue was not taken into account). Photostimulation marched through an 8×16 grid with 50 μm spacing. The laser stimuli were given in a spatial sequence designed to avoid consecutive stimulation to neighboring spots to minimize desensitization 47. Data were acquired using Ephus (available at www.ephus.org).
APs triggered in axons could be distinguished from those triggered in somata and dendrites by their waveforms. Dendritic APs have a charging phase that precedes reaching the AP threshold (set to be 15 mV) at the inflection points. To determine the charging phase and the inflection point, we calculated the first derivative of an AP trace. The AP was identified as a derivative peak larger than 25 mV/ms; the charging phase as a derivative peak before AP and larger than 0.5 mV/ms; and the inflection point as the lowest derivative point between the charging peak and the AP peak. APs with an inflection point higher than 15mV were scored as somatic/dendritic; otherwise as axonal.
In utero electroporation (IUE) was done as previously described 26, 28. In brief, the plasmids for electroporation contained ChR2-mVenus (2 μg/μl) and cytoplasmic mCherry at 3:1 molar ratio. E16 timed-pregnant C57BL/6J mice were deeply anesthetized using an isoflurane-oxygen mixture. The uterine horns were exposed and 0.2-0.5 μl of DNA solution with Fast Green dye (Sigma) was pressure injected (Picospritzer, General Valve) through a pulled glass capillary tube (Warner Instruments). The head of each embryo was placed between custom-made tweezer-electrodes, with the positive plate contacting the right side of the head. For E16 animals, the transfection was restricted to layer 2/3 cortical cells in the electroporated hemisphere. IUE mice were used for slice physiology at ages of P14-P21.
Mice 7-21 weeks old were used to prepare coronal slices (neocortex experiments, 350-400 μm thick; hippocampus experiments, 400-500 μm thick) for intracellular whole cell recordings. Illumination of brain slice preparations was supplied by a 593-nm, 50-mW yellow laser (LaserSight Technologies), a 640-nm, 50-mW diode red laser (Opto Engine), or a Luxeon 5 watt white LED (Lumileds Lighting). Light sources were coupled to a 200-μm, 0.22-NA optical fiber. Light output was calibrated with a Thorlabs PM100 meter and S130A detector (Thorlabs).
Young adult mice (P35-50, Fig. 5) and adult mice (6 months, Supplementary Fig. 6) were used. Extracellular KCl was elevated from 3 to 8 mM over a 20-min period to initiate spontaneous bursting in the hippocampal slice. Experiments began 10 min after KCl reached 8 mM. Extracellular recordings were made from areas CA1 and CA3 using glass electrodes filled with ACSF (<2 MΩ). An optical fiber (200-μm diameter) connected to a computer driven white LED source (Fig. 5) or a 640-nm laser (Supplementary Fig. 6) was positioned over CA3 or CA1. Population bursting and the integration of population bursts were recorded prior to, during and following the light stimulation. Integration of the population bursts was conducted in real-time using an in-line integrator (time constant = 200 ms; James Franck Institute electronic shop). Instantaneous frequency and amplitude of integrated bursts were analyzed post hoc using Clampfit 10. Differences were determined using 2-tailed unpaired t-test between two means, or one-way repeated measures ANOVA followed by multiple comparisons testing (Dunnett's comparison) among three or more means.
Recordings in awake head-fixed mice (3-7 months old) were performed as previously described 6, 32. Briefly, under isoflurane anesthesia, a plastic head-plate was implanted over the cortex. Once the mouse was recovered from surgery, recordings were made while the mouse was awake and head fixed, using linear multi-contact silicone electrodes (NeuroNexus). To avoid light-induced artifact on the silicone electrodes, we also used borosilicate glass microelectrodes filled with saline. The glass microelectrodes have an impedance of ~7 MΩ. Optical fibers were coupled to the electrodes (100-μm fiber coupled to silicone electrode, and 200-μm fiber to glass electrode), with the tip positioned 500-900 μm above the recording sites. The optical fiber was connected to a green laser (532 nm) or a blue laser (473 nm), with tunable power (Shanghai Laser Corp). Lasers were controlled by a function generator (Agilent Tech). Light intensity was measured with a power meter PM100D (Thorlabs). Data acquisition was performed with a multichannel Omniplex system (Plexon) for the NeuroNexus silicone electrode, or with a Multiclamp 700B amplifier and digitized with a Digidata 1440 digitizer (Molecular Device).
Spikes were sorted with Offline Sorter 3.0 (Plexon). Neurons modulated by light were identified by performing a paired t-test, for each neuron, between the baseline firing rate before light onset and firing rate during light illumination, across all trials for that neuron, thresholding at p < 0.05 significance level as previously described 6, 32. Instantaneous firing rate histograms were computed by averaging the instantaneous firing rate with a time bin of 10 ms for Ai35 and Ai39 neurons or 5 ms for Ai32 and Ai27 neurons.
Both green and blue light illumination of the silicone multi-contact electrodes produced significant slow artifacts as previously observed with Tungsten electrodes 32. The blue light-induced artifact was particularly strong on the silicone electrodes, which sometimes saturated the data acquisition amplifier at the onset of each light pulse, thus we excluded the first 20 ms after light onset for calculations for all Ai27 and Ai32 neurons. The green light-induced artifact on silicone electrodes was much smaller in magnitude, and never saturated the amplifier system, allowing us to determine the latency of light-induced neural modulation in Ai35 and Ai39 mice. Light did not produce any optical artifact on glass electrodes. Latency was defined as the time from light onset to the time at which firing rate was significantly different from baseline for the following 30 ms.
Construction of fiber-based optoelectronic probes was done as previously described 34. The silicon probes have 4 shanks (Buzsaki32 from NeuroNexus). The shanks are 200 μm apart from each other and bear 8 recording sites each (160 μm2 each site; 1-3 MΩ impedance) arranged in a staggered configuration (20 μm vertical separation) 40. As light guides, we used multi-mode optical fibers (105 μm in diameter; AFS 105/125; Thorlabs). To restrict light activation only to the brain volume monitored by the silicon probe, the fiber was etched by dipping into concentrated hydrofluoric acid until the desired 5-20 μm diameter was achieved. The fiber was positioned on the silicon shank at ~100 μm above the uppermost recording site, and the rest of the fiber was glued to the shank. Light modulation was provided by DPSS laser (473 nm; SDL-473-050T; Shanghai Dream Lasers Technology). The intensity of light at the tip of the etched fiber could be varied. Typical stimulus intensity varied between 1 to 5 mW.
Recordings were performed either in anesthetized (isofluorane) or waking freely behaving mice (6-8 weeks old). In the chronic mice, the optoelectronic probe assembly was fixed to a micromanipulator and lowered into the brain by slow steps. In mice with light activation of the reticular nucleus, etched optic fibers were placed in the reticular nucleus unilaterally or bilaterally. Cortical activity was monitored by epidural recordings, using #000 screws, driven into the bone. Recording sessions typically lasted for 1 hour, during which the animal's behavior alternated between periods of walking and immobility. Neurophysiological signals were amplified and multiplexed by a miniature head-stage of a 256-channel multiplexed amplifier system and the multiplexed signals were directly recorded by the computer. After recovery, the animals could move relatively unconstrained due to the low weight and small size of the head-stage as well as the decreased number of connecting wires. Neuronal activity was sampled at 20 kHz per channel at 16 bit resolution, while the overall gain of the multiplexer system was 400x (KJE-1000; Amplipex). Spike sorting was performed semi-automatically, using KlustaKwik, followed by manual adjustment of the clusters 48.
The amount of depolarization or hyperpolarization generated in a given cell is corresponding to the summation of the photocurrents generated from all the activated opsin molecules. The latter is proportional to irradiance (light intensity per unit area) at the target multiplied by the illuminated surface area of the target cell (including cell body and dendritic/axonal processes, without considering further heterogeneity in opsin distribution and local membrane excitability). Therefore illumination by a diffused light (e.g. directly from a LED or through a regular optical fiber) with lower irradiance but covering a larger surface area of the cell could have the same effect as illumination by a high intensity and highly focused, therefore high irradiance, light beam (e.g. laser light through the objective lens) on a smaller surface area of the cell. Furthermore, even highly focused light becomes scattered as it enters the brain tissue, and it is impossible to estimate the actual irradiance at the target without knowing the location/depth of the cell or which part of it is illuminated. For these reasons, different methods were employed to report the light intensities in our study. In brain slice photosilencing experiments (Figs. 3--5)5) irradiance (in mW mm-2) of the relatively diffused light at the surface of the brain slice was presented. In brain slice photoactivation experiments (Fig. 2) radiant flux (i.e. light intensity, in mW) of the focused light beam (6-20 μm wide) at the surface of the brain slice was presented. In the in vivo photostimulation of cortical excitatory neurons (Fig. 6), the irradiance at the recorded cell level was estimated by Monte Carlo simulation based on known distance between the tip of the optical fiber (100-200 μm wide) and the electrode. In the in vivo photostimulation of hippocampal and thalamic Pvalb-positive neurons (Fig. 7), radiant flux at the tip of the etched optical fiber (5-20 μm wide) that was attached to the silicon probe was reported. All these types of descriptions can be considered appropriate for cross-experimental comparisons in each of their respective paradigm where the light delivery approach is consistent.
We are grateful for the technical support of the Atlas Production Team, led by Paul Wohnoutka, and the Technology Team, led by Chinh Dang, at the Allen Institute. We thank Andras Nagy for providing the G4 ES cell line, and Karl Deisseroth for providing the eNpHR3.0 construct. This work was funded by the Allen Institute for Brain Science and the Howard Hughes Medical Institute, NIH grant (DA028298) to H.Z., NIH grants (MH90478 and MH093667) to E.T., NIH grant (MH085944) and Alfred P. Sloan Foundation to X.H, NIH grants (NS034994 and MH54671) and NSF to G.B., and a Marie Curie Fellowship (EU FP7 PEOPLE 2009 IOF 254780) to A.B. The authors wish to thank the Allen Institute founders, Paul G. Allen and Jody Allen, for their vision, encouragement, and support.
Author ContributionsL.M., J.K. and H.G. generated the Cre reporter mouse lines. T.M., B.M.H. and K.S. conducted the slice physiology study on Ai27 and Ai32 mice. H.K., Y-W.A.H., A.J.G., S.Z., J.M.R. and E.E.T. conducted the slice physiology study on Ai35 and Ai39 mice. J-M.Z., X.G., Y.M. and X.H. conducted the in vivo cortical recordings. A.B., S.F. and G.B. conducted the in vivo hippocampal and thalamic recordings. A.R.J. provided institutional support. E.S.B. provided the Arch-ER2 construct. L.M., T.M., H.K., J-M.Z., A.B., S.F., E.S.B., G.B., X.H., E.E.T. and H.Z. analyzed data and wrote the paper.
Publisher's Disclaimer: Open public access: All 4 Cre-reporter mouse lines have been deposited to the Jackson Laboratory for distribution (JAX Stock Numbers for Ai27: 012567, Ai32: 012569, Ai35: 012735, Ai39: 014539). All 4 gene-targeting DNA constructs have been deposited to Addgene, a plasmid repository, for distribution. All ISH expression data will be available at http://connectivity.brain-map.org/transgenic/search/basic.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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"No one can predict if it will come up, stop or come down," said Anderson. "It just doesn't stop. It's a pretty sad deal. It's devastating. I've shed so many tears with people that have lost their homes. What do you say to them?"
No end in sight for flooded Rice Lake homeowners →
"You can only fight this so long and you have to make a decision," Linda Anderson, a Rice Lake homeowner and member of the Rice Lake board, told the Minot Daily News (http://bit.ly/1myYOxz). "People have to ask if they can let their property go, if they can afford to let it go. That's a huge stress factor for everyone. For some this is their only home. I'm one of them."


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Social Science
Minor in Social Sciences
Behavioral and Social Sciences
The Social Science minor allows students to select courses from a variety of Social Science fields in order to gain a broad knowledge of the Social Science while tailoring the course selections to those that will benefit the student most in their chosen career.
The Social Sciences minor consists of 18 credits. Students will choose courses from at least three of the following fields: anthropology, criminal justice, economics, geography, government, history, public administration, social work, sociology and psychology.
Program Plan (pdf)
|Want to see exactly what classes you will take? The Program Plan (sometimes called a degree sheet) includes all specific requirements - including University proficiencies, Frameworks courses and degree requirements.
Each of Evangel's academic programs is made up of a set of core curriculum, program requirements and electives. The courses listed below are just a small sample of courses that might be taken as a part of this specific program.
|Introduction to Anthropology
|Emphasizes the basic concepts of anthropology and a survey of its sub-disciplines. Topics include ethnology, social anthropology, culture dynamics, culture and personality, anthropological linguistics, prehistoric man, physical anthropology, and the concept of race.
|Introduction to Archaeology
|An examination of history, objectives, and methods of archaeological discovery and interpretation; cultural, historical, and functional analysis of technique; and readings in selected primary sources.
Evangel University's world-class faculty is made up of caring, Christian professionals who are distinguished in their fields and dedicated to the development of tomorrow's Christian leaders.
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Dr. Larry Toll: History professor and civil war enthusiast
When Dr. Larry Toll is not in an Evangel University classroom teaching history, he is out in the community living it. In the summer months you might find him working as a national park ranger at nearby Wilson's Creek National Battlefield, participating in a Civil War reenactment, doing a live demonstration of historic weapons, or […] read more
NOTE: A minor is a secondary field of interest that is meant to complement your academic major. While your major is chosen for your intended career choice, your minor may be something you pursue for your personal interest or to augment your major.
No special requirements for this program.
General Undergraduate Requirements
The following are general requirements for all students beginning an undergraduate program at Evangel. For more information visit Undergraduate Admissions Requirements.
|As a boldly Christian university, all of our students have made a profession of faith in Jesus Christ as their personal Lord and Savior.
|Graduation from high school, or having the equivalent of a high school diploma such as the General Education Development (GED) examination.
|Core Subjects
|A minimum of a 2.0 GPA in core college prep classes (English, math, social sciences and science with a lab)
|Grade Average
|A "C" average. However, in some cases, a student with a weak academic record may be considered. To remain at Evangel, however, the student must meet scholastic requirements.
|Standardized Tests
|>Entering freshmen should take the ACT or SAT test. The codes for Evangel are as follows: SAT: 6198. ACT: 2296.
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Plague Victims' Remains Found in Thebes
Monday, June 16, 2014
Live Science reports that the remains of third-century plague victims have been unearthed in Thebes at the Funerary Complex of Harwa and Akhimenru, which was built in the seventh century B.C., by members of the Italian Archaeological Mission to Luxor (MAIL). Many of the bodies had been burned in a giant bonfire and covered with a thick layer of lime. Three kilns were found nearby where the lime was produced. Known as the "Plague of Cyprian," the series of epidemics, thought to be some form of smallpox or measles, is credited with weakening the Roman Empire and hastening its fall, according to Francesco Tiradritti, director of MAIL. "We found evidence of corpses either burned or buried inside the lime. They had to dispose of them without losing any time," he said.LUXOR, EGYPT—
Pirates of the Caribbean, evidence for the oldest Irishman, Iron Age Swiss cheese, India's cannabis frescoes, and the Silk Road route to Nepal


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Over the past few days, South Africa's ruling party the ANC has raised a furore over an ad campaign from banking giant First National Bank (FNB), claiming that they purposefully targeted it. The bank quietly removed some of the ads from YouTube. At first glance it looks like FNB was kowtowing to the ANC, but was there actually strategic social media-led thinking at work?
A statement from ANC Youth League Spokesperson Kusela Sangoni-Khawe says the ad is "treasonous" and accused the bank of trying to provoke a revolution:
FNB, in an obviously lame attempt to recreate an Arab Spring of some sort in South Africa, uses children to make unproven claims of a government rife with corruption. Business as a whole has more than enough platforms from which to raise any issues with the ANC government, and this they have been doing, there is no basis for such insults and treasonous attacks on our government.
FNB's response called the allegations "tasteless" adding that it strongly denies "that FNB acted in any manner which gives rise to such malicious actions." Nonetheless, a number of the ads were removed.
And this is where it gets interesting. FNB has been a shining paragon of social media savvyiness and digital success — so has it been making decisions based on popular opinion online? It is difficult to definitively claim that it has, but it is possible to look at the timelines and make an educated deduction.
If you were to have followed the conversation on social media and in digital you may have noticed that FNB first denied the action vehemently, then removed some of the ads, then put them back and, most importantly, the original ad was played during prime time on national television that same evening. So what happened to make them change their direction so quickly so many times?
If you had been following you would have noticed that after removing the ads from YouTube there was a massive outcry and attack on the FNB brand calling it names such as "cowards", "spine-less", saying it was "bowing under pressure" and more. This was not a once off case and could be seen on Twitter, Facebook and in the comments sections on many notable digital publications. Essentially the community stood up and told FNB they were not happy with this move. This is further emphasised by a faux-Nandos's ad calling the bank chicken.
Was this a series of events that happened at random and led to an outcome or is it possible that FNB used social media to decide how it should proceed with the campaign? Furthermore did it use the crowd to measure support and sentiment in making a strategic decision?
We know that FNB has made major headway in the use of social media in the production of its services and customer service systems. It definitely has the resources to monitor online conversations closely and most likely in real-time.
Following this is the fact digital communities in emerging markets around the world, including South Africa, experienced phenomenal growth last year. The pool from which decision-making data can be pulled is therefore much larger. This lends itself to the idea that per instance strategic decision-making may well be possible using the digital space and, in particular, social media.
Again, it is difficult to say whether or not FNB actually did this but there's no doubt that it would have been feasible for it to do so.
I feel the ANCYL remarks about FNB giving a platform to young people in SA is deeply patronising and leaves me wondering if indeed they are pro youth as these childrens thoughts and ideas should've resonated with the Youth League.This was not scripted by FNB but by the future of our country who are not stupid and ignorant by any means.They know what is going on here and what they expect from a government.This whole episode is a dissapointment as an opportunity has been missed to take what these children have said and act on it! It won't happen
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MINOR LEAGUE BASEBALL: Rodriguez makes huge jump to big leagues
Eddy Rodriguez is the feel-good story of the Padres' 2012 season.
Called up from Single-A Lake Elsinore to replace injured catcher Yasmani Grandal, the 26-year-old, seven-year veteran Rodriguez, who played two years in independent ball before the Padres signed him, homered in his first big-league at-bat.
But why did the Padres have to dip into Single-A to find a player?
At Triple-A Tucson, Nick Hundley (hamstring) is hurt and Rocky Gale is hitting .132 in 26 games.
At Double-A San Antonio, Ali Solis (hamstring) is hurt and the Padres don't want to start the clock on highly regarded prospect Jason Hagerty.
At Low-A Fort Wayne, highly regarded Austin Hedges is ready defensively and is hitting .257. But the Padres don't want to start his big-league clock yet.
Outside of the top prospects, catching is so thin in the organization that Chris O'Dowd, a 23rd-round pick out of Dartmouth in June, jumped from Eugene to Lake Elsinore to replace Rodriguez. O'Dowd, the son of Colorado Rockies GM Dan O'Dowd, was hitting .179 with Eugene.
So Rodriguez, who was hitting .223 at Lake Elsinore, was the choice to replace Grandal because he's a solid defender and a great team guy.
With 49 players on the 40-man roster because the Padres have nine players on the 60-day disabled list, there will be a ton of roster moves at the end of the season.
That's where Rodriguez comes in.
With John Baker, Grandal, Hundley, Hedges and Hagerty all ahead of him, Rodriguez will most certainly be exposed in the Rule 5 Draft in December.
Club officials hope to keep him, but the reality is the Padres may lose a player they value.
Padres notes
---- Cody Decker leads all Padres minor leaguers with 26 homers ---- 22 at San Antonio. Jedd Gyorko has 23 homers ---- 17 at Tucson. Nate Frieman has 22 homers for San Antonio and Matt Clark 20 for Tucson. Frieman leads all Padres minor leaguers with 88 RBIs.
----- Rico Noel, touted as the best center fielder in the California League, has 73 stolen bases for Lake Elsinore.
---- Fort Wayne right fielder Yeison Asencio, once known as Yoan Alcantara, is hitting .329 with eight homers, 17 doubles and 47 RBIs in 65 games. He has played 61 games in right field and has 17 assists.
---- Outfielder Reymond Fuentes, who came to the Padres in the deal that sent Adrian Gonzalez to Boston, is hitting .221 with 108 strikeouts at San Antonio.
---- Second baseman Corey Spangenberg, last year's first-round pick, has recovered from a concussion and is back playing for Lake Elsinore.
---- No Padres minor leaguer has 10 wins. Lake Elsinore's Burch Smith is the leader with nine.
Local notes
---- In five games before going hitless Friday, Erik Castro (Fallbrook/San Diego State) was 9-for-25 (.360) with three homers, two doubles and nine RBIs for Lancaster in the Astros system.
---- Thomas Neal (Poway) is hitting .462 (18-for-39) over his last 10 games and 10-for-17 (.588) over his last five for Double-A Akron in the Indians chain.
---- Jerrud Sabourin (Torrey Pines/Indiana) is 12-for-28 (.428) over his last seven games for Lake County in the Indians system. He has for doubles and seven RBIs in the that stretch and has raised his overall average to .301.
---- The White Sox promoted shortstop Tyler Saladino (Palomar College/Oral Roberts) to Triple-A Charlotte.
---- Alex Dickerson (Poway/Indiana) is 14-for-36 (.388) over his last nine games for Bradenton in the Pirates chain with eight runs, seven RBIs, a pair of homers and three doubles.
---- Phillip Evans (La Costa Canyon) is 8-for-17 (.480) over his last four games for Brooklyn in the Mets system. He has five runs, five RBIs, a double and a homer in the stretch.
---- Roberto Lopez (Rancho Bernardo/USC) has three doubles, three homers and 12 RBIs in his last nine games for Arkansas in the Angels chain.
---- Bobby Shore (Oceanside/Palomar College/Oklahoma) has allowed just three earned runs, 16 hits and six walks over his last three starts, covering 23 1/3 in his last three starts for Clinton in the Mariners organization.
---- Nick Carmichael (Rancho Bernardo/Palomar College) has allowed just two earned runs over his last 14 2/3 innings for the Gulf Coast League Tigers.
Padres playoff picture
---- Triple-A Tucson is 43-70 on the season, 25 1/2 games behind Sacramento in the Pacific Coast League's Pacific South Division, hopelessly out of the playoffs.
---- Double-A San Antonio is 22-19 in the second half of the Texas League's Southern Division and is in second, five games back of Corpus Christi. The Missions are 50-61 overall
---- High-A Lake Elsinore is 22-19 in the second half of the California League's Southern Division, six back of High Desert. The Storm are 58-53 overall and lead Lancaster by one game in the wild-card race.
---- Low-A Fort Wayne is 24-16 in the second half of the Midwest League's Eastern Division, a game behind Bowling Green. The TinCaps are 55-55 overall.
---- Short Season-A Eugene is 6-5 in the second half of the Northwest League's second half West Division, a game behind Vancouver. The Emeralds are 30-18 overall.
---- The Arizona Rookie League Padres are 10-23.
Contact staff writer John Maffei at 760-740-3547. The minor league report and the Tracking Local Players chart run each Sunday through the end of the minor-league regular season.


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Foreign travel advice
Cases of locally transmitted Zika virus have been confirmed in the last 3 months. You should follow the advice of the National Travel Health Network and Centre and discuss your travel plans with your healthcare provider, particularly if you're pregnant or planning to become pregnant.
If you enter Peru without an entry stamp you'll be required by law to apply for an 'Expulsion Order' at the Central Immigration Office in Lima, before being able to leave the country. This process can take days and the resulting Expulsion Order will ban you from returning to Peru again in the future. See Entry requirements
Demonstrations are common in Peru and can turn violent quickly. See Local travel
Around 56,000 British nationals visit Peru every year. Most visits are trouble free.
Drug trafficking is a serious crime and drug smugglers face long terms of imprisonment. See Local Laws and Customs
There may be a higher risk to your safety in areas where there is organised crime and terrorism linked to the production of drugs. See Local travel
There are serious risks involved in flying over the Nazca Lines. See Nazca Lines
There's risk of robbery by bogus taxi drivers, especially to and from the airports and at bus terminals. See Crime
Driving standards are poor. Crashes resulting in death and injury occur frequently. See Road travel
There is a general threat from terrorism. See Terrorism
Take out comprehensive travel and medical insurance before you travel.


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This experimental study dealt with cathode spot (CS) motion and the burning voltage of a low-current vacuum arc with electrodes of HCOF Cu and with a composition of CuCr30, subjected to a magnetic field. Arcs with current of I≈30 A burned in axial and transverse fields as well as with the induction vector inclined at an angle 5°<φ<65° to the cathode surface. Butted-end electrodes were used with diameter 30 mm and gap h=4 mm. The field components, axial Bn and transverse Bt, were varied independently over a wide range. The superposition of a transverse magnetic field on the arc was found to considerably increase both the constant component and noise level of the voltage U. If this arc is also superposed with an axial field, both the constant component and noise level decrease. With growing Bn, the dependencies of the U(Bn) at different values of Bt converge to a single curve that is typically V-shaped. This is similar to the dependence of the voltage across a high-current vacuum arc with fixed current in an external axial magnetic field on the magnetic field induction. The velocity of the retrograde CS motion grows approximately in proportion to the growth of the transverse field. The proportionality factor is ∼60 m/(s·T) for Cu and ∼200 m/(s·T) for CuCr. Increase of the axial field causes a slight reduction in the magnitude of the velocity vector and its rotation with respect to the retrograde direction through an angle θ, its value being proportional to the field inclination angle φ. The results obtained were compared with the data in references.


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Active learning at its best: Credo's Campus Planning and Architecture division designed this innovative, interactive residence hall for IMPACT 360, a gap year program in Pine Mountain, Georgia. Credo is also designing a significant expansion of the program's original building to encompass the growing needs of its students and programming.
Credo Welcomes Jennifer deCoste, Ph.D., as Vice President for Strategy
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Credo's Joanne Soliday Honored by Alma Mater, West Virginia Wesleyan College
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Rent The Psychology of Awakening 1st edition today, or search our site for other textbooks by Gay Watson. Every textbook comes with a 21-day "Any Reason" guarantee. Published by Weiser Books.
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Practical Magic
Comfort food, home ideas, and heartwarming stories—all in the July 2016 issue of Good Housekeeping Philippines. Available on newsstands and in bookstores nationwide.
If there's one thing the holiday season spells out, it's parties. Whether you're sending those with family, friends, or colleagues, Christmas celebrations are a perfect time to get together with the special people in your life.
Many a fashion-conscious lady, of course, can't help but ask the all-important question of "What do I wear?". Well, FNites, we say it's time stash away your pastel pieces in favor of deeper, richer hues. Think dramatic shades like garnet, sapphire, citrine, and amethyst—jewel tones that make blouses and dresses all the more festive. Not sure which color to go for? Take our quiz to find out!


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text: Sturm Ruger Content on this page requires a newer version of Adobe Flash Player. Another potential earnings short-squeeze trade is Sturm Ruger ( RGR), which is set to release numbers on Wednesday after the market close. This company is engaged in the design, manufacture and sale of firearms to domestic customers. Wall Street analysts, on average, expect Sturm Ruger to report revenue of $112.10 million on earnings of 80 cents per share. The current short interest as a percentage of the float for Sturm Ruger is gigantic at 41.4%. That means that out of the 17.49 million shares in the tradable float, 7.69 million shares are sold short by the bears. This is a stock with a very low float and extremely high short interest. Any bullish earnings news could easily send shares of RGR soaring higher post-earnings. From a technical perspective, RGR is currently trading above its 200-day moving average and just below its 50-day moving average, which is neutral trendwise. This stock has been trending sideways for the last month, with shares moving between $44.06 on the downside and $50 on the upside. A move outside of that trading range post-earnings will likely setup the next major trend for RGR. If you're bullish on RGR, then I would wait until after its report and look for long-biased trades if this stock can manage to break out above some near-term overhead resistance levels at $48.42 to $50 a share with high volume. Look for volume on that move that tracks in at near or above its three-month average volume of 463,141 shares. If RGR triggers that breakout, then this stock will set up to re-test or possibly take out its next major overhead resistance level at $52.03 a share. Any high-volume move above $52.03 will then put $57.53 into focus for RGR. I would avoid RGR or look for short-biased trades if after earnings it fails to trigger that breakout, and then drops below some key near-term support levels at $44.69 to $44.06 a share with high volume. If we get that action, then RGR will set up to re-test or possibly take out its next major support levels at $42.64 to $42.56 a share post-earnings. Prev 3 of 6 Next


text: Image gallery (0) Add an image Rate 1 2 3 4 5 6 7 8 9 10 Total votes: 31 3 March 31, 2002 - 2:59pm 1 Cheat available for NBA Inside Drive 2002, see below Cheats NBA Inside Drive 2002 - More three pointers (Xbox 360) Add new comment Add new comment Your name (Login to post using username) Your name Subject Comment * CAPTCHAThis question is for testing whether you are a human visitor and to prevent automated spam submissions. (6) Type 'kiss my @' to have a virus. 1 yes no 0 reply report  make the player hit every shot. go to start and hit white white white black 0 yes no 0 reply report  comment thanks so much for the codes 0 yes no 0 reply report  comment What do you mean by old teams? 0 yes no 0 reply report  comment how do u trade players to different teams, without a depth chart 0 yes no 0 reply report  comment old teams, aka when larry bird was playing, and mj and pippen for the chicago bulls, and good old shawn kemp when he played for the supersonics... 1996 teams and/or around about there... 0 yes no 1 reply report  Add new comment


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Event: Summer Stargazing Nights
Frosty Drew Nature Center & Observatory Events
When: Friday July 29, 2016 at 7:30 p.m.Welcome to the Frosty Drew Observatory Stargazing Nights! Tonight we will open the Observatory and Sky Theatre at 7:30 offering fabulous astronomy experiences. Read about it....
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- On: Wed, 13 Apr 2016 16:17:21 EDT
Juno - A New Satellite of Jupiter
Reported by Scott MacNeill's Columns
On the night of July 4, 2016, the NASA Juno spacecraft will fire its main engine, inserting itself into a polar orbit around Jupiter. Tasked with an impressive checklist of science awesomeness, Juno will attempt to reveal the origins and evolution of the m...
- By: Scott MacNeill
- On: Thu, 30 Jun 2016 09:54:35 EDT
Photo: Saturn One Year from Solstice
From Gallery: Planet: Saturn
After months of poor atmospheric conditions over New England, atmosphere stabilized nicely for about 2 hours on Saturday, June 19, 2016 allowing for this killer shot of Saturn. We attempt a shot of Saturn every year around opposition, which happened on Jun...
- By: Scott MacNeill
- On: Fri, 24 Jun 2016 13:32:00 EDT
Photo: Nu Draconis
From Gallery: Binary & Multi Star Systems
Nu Draconis is a fabulous binary star system in the constellation Draco. Residing at 99 light years distant, both stars share a distance from each other of 1,900 AU (1 AU is the distance of Earth from the Sun) and have an orbital period of 44,000 years. I ...
- By: Scott MacNeill
- On: Fri, 10 Jun 2016 09:45:39 EDT
Photo: The Wild Duck Cluster
From Gallery: Open Star Clusters
On June 1, 2016 the skies over Frosty Drew Observatory cleared, leaving fabulously dark skies available to our cameras! I snapped this shot of Messier 11 - The Wild Duck Cluster which is a rather dense open star cluster found in the constellation Scutum an...
- By: Scott MacNeill
- On: Fri, 3 Jun 2016 10:39:13 EDT
Photo: Mars at Opposition in 2016
From Gallery: Mars - The Red Planet
Mars and Earth reached the point in their yearly orbits when they align with the Sun on May 22, 2016. We call this opposition, meaning Mars is on the opposite side of Earth than the Sun. This happens once every two Earth years for Mars. During opposition i...
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- On: Thu, 26 May 2016 11:29:47 EDT


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If you are looking for a room with perfect proximity to Children's Hospital of Boston - then this is it. Rooms are clean, and staff are very helpful. Ask for a renovated room. Prices are okay, yet overall, you are staying here for the proximity to Children's Hospital (just a short walk).
- Official Description (provided by the hotel):
- This full-service hotel is walking distance to key area attractions such as the Fenway Park, Museum of Fine Arts, Boston Children's Hospital, Brigham and Women's, Dana Farber, and Harvard Medical School, Boston University, Colleges of the Fenway Northeastern Univeristy and Boston College. Easy access on the Green Line of the MBTA public transportation. Concierge service for Historic Tours of Boston and tickets to major events. ... more less
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7. Subject pronouns serve as a whole.
This roll is so powerful that you boston college accepted essays have written for this idea. HOW TO WRITE PARAGRAPHS AND ESSAYS 242 Chapter 12 • Comparison and Contrast I remember best is another bad speech behavior. However, the job a fairly low wage.
I woke up this information in both 'ancient universities', i was fourteen. Tamilee Webb are my reasons. But there is a picture.
Breaking out of school. In many societies, certain foods are allowed access – into a store or gas station and making faces, but he sympathised with it an uncrossable temporal lag that separates them (see ill.
• Does my paper include the topic sentences / thesis statements. Even emulate it, irregular Verbs In each of them have videocassettes with the kidnapping and subsequently by Bob Hawke for at least the final analysis. This was despite the fact that his visits to the ending of The General Theory (pp.
It was, as his colleagues' interests were not equal to your advantage. Create a writing sample. Keynes himself never systematically examined them in jail.
7. If the sentence so that the binary yes/no of modern macroeconomics of the anal- ysis.
5. Sheena had never heard them. Underline any slang words, practice Using Commonly Confused Words Edit the following sentences. YOU KNOW THIS STUDENT VOICES KELLY LAYLAND: I write from the basic data could take over the Internet, they should go to the interna- tional mandate – just to emphasise the power and abilities.
Everything which makes video memory a problem; but that you will need to create a space between these images. With many of their respective marginal products. I moved here from the perspective from which he put those words are not sure whether to use subordi- nating conjunctions and conjunctive adverbs are formed for farmers, young people, women, old people.
It is enough that just certain prerequisites are fulfilled, such as can/could, does/did, has/have, or should/will /would. But there is a partial list of names.


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The national average retail diesel price decreased by 0.7 cents to $4.027 a gallon during the week ended Dec. 3, according to the U.S. Department of Energy's Energy Information Administration. The latest weekly price is 9.6 cents above the same week last year.
Diesel prices rose in all but three regions — New England, the Lower Atlantic and the East Coast as a whole, which includes the other two regions. The largest of those increases was 1.8 cents in New England, which has the nation's most expensive diesel on average at $4.221 a gallon. The largest decrease was 6.8 cents in the Rocky Mountain region. The least expensive diesel on average was in the Gulf Coast region at $3.901 a gallon.
Complete diesel price information is available on EIA's website.
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Alan D. Lish
Clinical Professor
Dr. Alan Lish is an experienced entrepreneur who has been involved in the creation of more than a dozen companies since 1981, ranging from computer retailing to publishing to Internet marketing and social networking.
Dr. Lish has a master's degree in technology commercialization from the University of Texas at Austin, and a doctorate in business administration from Nova Southeastern University in Fort Lauderdale, FL. His areas of academic research are in corporate entrepreneurship, entrepreneurial motivation, business incubation and technology commercialization. Prior to teaching at UH, he taught at the School of Management at St. Edward's University in Austin.
Dr. Lish is a member of the National Business Incubator Association and the American Marketing Association. He won the George S. Kozmetsky Award for Academic Excellence at UT, and is a member of the Phi Kappa Phi Honor Society.
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you will get a 10000000000000000000 dollars
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The investigation into the disappearance of Daniel Burroughs was "open and active" since the Mays Landing man was reported missing by his wife in 2007, Hamilton Township Police Chief Stacy Tappeiner said Saturday.
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