Comprehensive knowledge of the brain’s wiring diagram is fundamental for understanding how the nervous system processes information at both local and global scales. However, with the singular exception of the C. elegans microscale connectome, there are no complete connectivity data sets in other species. Here we report a brain-wide, cellular-level, mesoscale connectome for the mouse. The Allen Mouse Brain Connectivity Atlas uses enhanced green fluorescent protein (EGFP)-expressing adeno-associated viral vectors to trace axonal projections from defined regions and cell types, and high-throughput serial two-photon tomography to image the EGFP-labelled axons throughout the brain. This systematic and standardized approach allows spatial registration of individual experiments into a common three dimensional (3D) reference space, resulting in a whole-brain connectivity matrix. A computational model yields insights into connectional strength distribution, symmetry and other network properties. Virtual tractography illustrates 3D topography among interconnected regions. Cortico-thalamic pathway analysis demonstrates segregation and integration of parallel pathways. The Allen Mouse Brain Connectivity Atlas is a freely available, foundational resource for structural and functional investigations into the neural circuits that support behavioural and cognitive processes in health and disease.
Nervous systems are composed of various cell types, but the extent of cell type diversity is poorly understood. Here, we construct a cellular taxonomy of one cortical region, primary visual cortex, in adult mice based on single cell RNA-sequencing. We identify 49 transcriptomic cell types including 23 GABAergic, 19 glutamatergic and seven non-neuronal types. We also analyze cell-type specific mRNA processing and characterize genetic access to these transcriptomic types by many transgenic Cre lines. Finally, we show that some of our transcriptomic cell types display specific and differential electrophysiological and axon projection properties, thereby confirming that the single cell transcriptomic signatures can be associated with specific cellular properties.
This Matters Arising paper is in response to Guo et al (2013) in Neuron. See also the Matters Arising by Eckler et al. published concurrently. Using genetic fate-mapping with Cux2-Cre and Cux2-CreERT2 mice we demonstrated that the neocortical ventricular zone (VZ) contains radial glial cells (RGCs) with restricted fate potentials (Franco et al., 2012). Using the same mouse lines, Guo et al. (2013) concluded that the neocortical VZ does not contain lineage restricted RGCs. We now show that the recombination pattern in Cux2-Cre/CreERT2 mice depends on genetic background and breeding strategies. We provide evidence that Guo et al. likely reached different conclusions because they worked with transgenic sublines with drifted transgene expression patterns. In Cux2-Cre and Cux2-CreERT2 mice that recapitulate the endogenous Cux2 expression pattern, the vast majority of fate-mapped neurons express Satb2 but not Ctip2, confirming that a restricted subset of all neocortical projection neurons belongs to the Cux2 lineage.
An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems, and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.
Optogenetics provides a unique approach to remotely manipulate brain activity with light. Reaching the degree of spatiotemporal control necessary to dissect the role of individual cells in neuronal networks, some of which reside deep in the brain, requires joint progress in opsin engineering and light sculpting methods. Here we investigate for the first time two-photon stimulation of the red-shifted opsin ReaChR. We use two-photon (2P) holographic illumination to control the activation of individually chosen neurons expressing ReaChR in acute brain slices. We demonstrated reliable action potential generation in ReaChR-expressing neurons and studied holographic 2P-evoked spiking performances depending on illumination power and pulse width using an amplified laser and a standard femtosecond Ti:Sapphire oscillator laser. These findings provide detailed knowledge of ReaChR's behavior under 2P illumination paving the way for achieving in depth remote control of multiple cells with high spatiotemporal resolution deep within scattering tissue.
optogenetics; 2-photon excitation; computer generated holography; opsin; action-potential generation; neuroscience; cortex
We report a method to facilitate single cell, image-guided experiments including in vivo electrophysiology and electroporation. Our method combines 3D image data acquisition, visualization and on-line image analysis with precise control of physical probes such as electrophysiology microelectrodes in brain tissue in vivo. Adaptive pipette positioning provides a platform for future advances in automated, single cell in vivo experiments.
Intracellular Ca2+ signaling is considered important for multiple astrocyte functions in neural circuits. However, mice devoid of inositol triphosphate type 2 receptors (IP3R2) reportedly lack all astrocyte Ca2+ signaling, but display no neuronal or neurovascular deficits, implying that astrocyte Ca2+ fluctuations play no role(s) in these functions. An assumption has been that loss of somatic Ca2+ fluctuations also reflects similar loss within astrocyte processes. Here, we tested this assumption and found diverse types of Ca2+ fluctuations within astrocytes, with most occurring within processes rather than in somata. These fluctuations were preserved in IP3R2−/− mice in brain slices and in vivo, occurred in endfeet, were increased by G-protein coupled receptor activation and by startle-induced neuromodulatory responses. Our data reveal novel Ca2+ fluctuations within astrocytes and highlight limitations of studies that used IP3R2−/− mice to evaluate astrocyte contributions to neural circuit function and mouse behavior.
The structural organization of neural circuits is strongly influenced by experience, but the underlying mechanisms are incompletely understood. We found that, in the developing dentate gyrus (DG), excitatory drive promotes the somatic innervation of principal granule cells (GCs) by parvalbumin (PV)-positive basket cells. By contrast, presynaptic differentiation of GCs and interneuron sub-types that inhibit GC dendrites is largely resistant to loss of glutamatergic neurotransmission. The networks of PV basket cells in the DG are regulated by vesicular release from projection entorhinal cortical neurons and, at least in part, by NMDA receptors in interneurons. Finally, we present evidence that glutamatergic inputs and NMDA receptors regulate these networks through a presynaptic mechanism that appears to control the branching of interneuron axons. Our results provide insights into how cortical activity tunes the inhibition in a subcortical circuit, and reveal new principles of interneuron plasticity.
GABAergic synaptic transmission plays an important role in resetting and synchronizing circadian rhythms in the suprachiasmatic nucleus (SCN). Although the circadian modulation of intrinsic membrane currents and biochemical signaling have been examined in the SCN, the modulation of specific synaptic pathways within the SCN is unexplored. In addition, little is known about the functional properties of these pathways, including which ones involve GABAA receptors (GABAA-Rs). In brain slices obtained from mice, we examined synaptic responses originating from the SCN neurons expressing vasoactive intestinal peptide (VIP+ neurons). Focusing on the local projection within the ventromedial SCN, we found that VIP+ afferents provided input onto 49% of neurons with a preference for VIP-negative (VIP−) neurons. Responses were mediated by GABAA-Rs. The projection was sparsely connected and preferentially targeted a subset of SCN neurons unrelated to postsynaptic VIP expression. For most aspects of VIP+ network output, there was no circadian regulation. Excitability and spontaneous firing of the presynaptic VIP+ neurons were unchanged between day and night, and their network connectivity and synaptic function up through the evoked synaptic conductance were also unchanged. On the other hand, VIP+ input onto VIP− neurons became less inhibitory at night suggesting a postsynaptic alteration in the coupling of GABAA-R conductances to action potential firing. These data suggest that components of the VIP network and its synaptic output up through GABAA-R opening are invariant during the circadian cycle, but the effect on action potential firing is modulated by postsynaptic processes occurring after GABAA-R channel opening.
circadian; GABA; network; SCN; synapse; VIP
Understanding how behavior emerges from brain electrical activity is one of the ultimate goals of neuroscience. To achieve this goal we require methods for large-scale recording of the electrical activity of specific neuronal circuits. A very promising approach is to use optical reporting of membrane voltage transients, particularly if the voltage reporter is genetically targeted to specific neuronal populations. Targeting in this way allows population signals to be recorded and interpreted without blindness to neuronal diversity. Here, we evaluated the voltage-sensitive fluorescent protein, VSFP Butterfly 2.1, a genetically encoded voltage indicator (GEVI), for monitoring electrical activity of layer 2/3 cortical pyramidal neurons in mouse brain slices. Standard widefield fluorescence and two-photon imaging revealed robust, high signal-to-noise ratio read-outs of membrane voltage transients that are predominantly synaptic in nature and can be resolved as discrete areas of synaptically connected layer 2/3 neurons. We find that targeted expression of this GEVI in the cortex provides a flexible and promising tool for the analysis of L2/3 cortical network function.
Cortex; genetically encoded voltage indicator; L2/3; optogenetics; voltage imaging
Elucidating the genetic control of cerebral cortical (pallial) development is essential for understanding function, evolution, and disorders of the brain. Transcription factors (TFs) that embryonically regulate pallial regionalization are expressed in gradients, raising the question of how discrete domains are generated. We provide evidence that small enhancer elements active in protodomains integrate broad transcriptional information. CreERT2 and GFP expression from 14 different enhancer elements in stable transgenic mice allowed us to define the first comprehensive regional fate map of the pallium. We explored transcriptional mechanisms that control the activity of the enhancers using informatics, in vivo occupancy by TFs that regulate cortical patterning (CoupTFI, Pax6 and Pbx1), and analysis of enhancer activity in Pax6 mutants. Overall, the results provide novel insights into how broadly expressed patterning TFs regulate the activity of small enhancer elements that drive gene expression in pallial protodomains that fate map to distinct cortical regions.
The climbing fiber input to the cerebellar cortex is thought to provide instructive signals that drive the induction of motor skill learning. We found that optogenetic activation of Purkinje cells, the sole output neurons of the cerebellar cortex, can also drive motor learning in mice. This dual control over the induction of learning by climbing fibers and Purkinje cells can expand the learning capacity of motor circuits.
Social behaviors, such as aggression or mating, proceed through a series
of appetitive and consummatory phases1 that are associated with increasing levels of
arousal2. How such
escalation is encoded in the brain, and linked to behavioral action selection,
remains an important unsolved problem in neuroscience. The ventrolateral
subdivision of the murine ventromedial hypothalamus (VMHvl) contains neurons
whose activity increases during male-male and male-female social encounters.
Non-cell type-specific optogenetic activation of this region elicited attack
behavior, but not mounting3. We
have identified a subset of VMHvl neurons marked by the estrogen receptor 1
(Esr1), and investigated their role in male social behavior. Optogenetic
manipulations indicated that Esr1+ (but not Esr1-)
neurons are sufficient to initiate attack, and that their activity is
continuously required during ongoing agonistic behavior. Surprisingly, weaker
optogenetic activation of these neurons promoted mounting behavior, rather than
attack, towards both males and females, as well as sniffing and close
investigation (CI). Increasing photostimulation intensity could promote a
transition from CI and mounting to attack, within a single social encounter.
Importantly, time-resolved optogenetic inhibition experiments revealed
requirements for Esr1+ neurons in both the appetitive
(investigative) and the consummatory phases of social interactions. Combined
optogenetic activation and calcium imaging experiments in
vitro, as well as c-Fos analysis in vivo, indicated
that increasing photostimulation intensity increases both the number of active
neurons and the average level of activity per neuron. These data suggest that
Esr1+ neurons in VMHvl control the progression of a
social encounter from its appetitive through its consummatory phases, in a
scalable manner that reflects the number or type of active neurons in the
Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain.
Large three-dimensional images are commonly generated through biological experimentation. Here the authors report software tools for exploration of three-dimensional images along with applications to assist in imaging, microsurgery, visualization and annotation of large image data sets.
The Chrna5 gene encodes the α5 nicotinic acetylcholine receptor subunit, an “accessory” subunit of pentameric nicotinic receptors, that has been shown to play a role in nicotine-related behaviors in rodents and is genetically linked to smoking behavior in humans. Here we have used a BAC transgenic mouse line, α5GFP, to examine the cellular phenotype, connectivity, and function of α5-expressing neurons. Although the medial habenula (MHb) has been proposed as a site of α5 function, α5GFP is not detectable in the MHb, and α5 mRNA is expressed there only at very low levels. However, α5GFP is strongly expressed in a subset of neurons in the interpeduncular nucleus (IP), median raphe/paramedian raphe (MnR/PMnR), and dorsal tegmental area (DTg). Double-label fluorescence in situ hybridization reveals that these neurons are exclusively GABAergic. Transgenic and conventional tract tracing show that α5GFP neurons in the IP project principally to the MnR/PMnR and DTg/interfascicular dorsal raphe, both areas rich in serotonergic neurons. The α5GFP neurons in the IP are located in a region that receives cholinergic fiber inputs from the ventral MHb, and optogenetically assisted circuit mapping demonstrates a monosynaptic connection between these cholinergic neurons and α5GFP IP neurons. Selective inhibitors of both α4β2- and α3β4-containing nicotinic receptors were able to reduce nicotine-evoked inward currents in α5GFP neurons in the IP, suggesting a mixed nicotinic receptor profile in these cells. Together, these findings show that the α5-GABAergic interneurons form a link from the MHb to serotonergic brain centers, which is likely to mediate some of the behavioral effects of nicotine.
The dentate gyrus (DG), in addition to its role in learning and memory, is increasingly implicated in the pathophysiology of anxiety disorders. Here, we show that, dependent on their position along the dorso-ventral axis of the hippocampus, DG granule cells (GCs) control specific features of anxiety and contextual learning. Using optogenetic techniques to either elevate or decrease GC activity, we demonstrate that GCs in the dorsal DG control exploratory drive and encoding, not retrieval, of contextual fear memories. In contrast, elevating the activity of GCs in the ventral DG has no effect on contextual learning but powerfully suppresses innate anxiety. These results suggest that strategies aimed at modulating the excitability of the ventral DG may be beneficial for the treatment of anxiety disorders.
Significant advances in circuit-level analyses of the brain require tools that allow for labeling, modulation of gene expression, and monitoring and manipulation of cellular activity in specific cell types and/or anatomical regions. Large-scale projects and individual laboratories have produced hundreds of gene-specific promoter-driven Cre mouse lines invaluable for enabling genetic access to subpopulations of cells in the brain. However, the potential utility of each line may not be fully realized without systematic whole brain characterization of transgene expression patterns. We established a high-throughput in situ hybridization (ISH), imaging and data processing pipeline to describe whole brain gene expression patterns in Cre driver mice. Currently, anatomical data from over 100 Cre driver lines are publicly available via the Allen Institute's Transgenic Characterization database, which can be used to assist researchers in choosing the appropriate Cre drivers for functional, molecular, or connectional studies of different regions and/or cell types in the brain.
Cre driver mice; genetic tools; anatomical characterization; in situ hybridization; neuronal cell types
Odor stimulation evokes complex spatiotemporal activity in the olfactory bulb, suggesting that the identity of activated neurons as well as the timing of their activity convey information about odors. However, whether and how downstream neurons decipher these temporal patterns remains debated. We addressed this question by measuring the spiking activity of downstream neurons while optogenetically stimulating two foci in the olfactory bulb with varying relative timing in mice. We found that the overall spike rates of piriform cortex neurons were sensitive to the relative timing of activation. Posterior piriform cortex neurons showed higher sensitivity to relative input times than neurons in the anterior piriform cortex. In contrast, olfactory bulb neurons rarely showed such sensitivity. Thus, the brain can transform a relative time code in the periphery into a firing-rate-based representation in central brain areas, providing evidence for the relevance of relative time-based code in the olfactory bulb.
Although there have been major advances in elucidating the functional biology of the human brain, relatively little is known of its cellular and molecular organization. Here we report a large-scale characterization of the expression of ~1,000 genes important for neural functions, by in situ hybridization with cellular resolution in visual and temporal cortices of adult human brains. These data reveal diverse gene expression patterns and remarkable conservation of each individual gene’s expression among individuals (95%), cortical areas (84%), and between human and mouse (79%). A small but substantial number of genes (21%) exhibited species-differential expression. Distinct molecular signatures, comprised of genes both common between species and unique to each, were identified for each major cortical cell type. The data suggest that gene expression profile changes may contribute to differential cortical function across species, in particular, a shift from corticosubcortical to more predominant corticocortical communications in the human brain.
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 universal behaviors, such as sensation, movement, cognition, and emotion, arise from the interactions of specific cells that are present within intricate neural networks in the brain. Dissection of such complex networks has typically relied on disturbing the activity of individual gene products, perturbing neuronal activities pharmacologically, or lesioning specific brain regions, to investigate the network’s response in a behavioral output. Though informative for many kinds of studies, these approaches are not sufficiently fine-tuned for examining the functionality of specific cells or cell classes in a spatially or temporally-restricted context. Recent advances in the field of optogenetics now enable researchers to monitor and manipulate the activity of genetically defined cell populations with the speed and precision uniquely afforded by light. Transgenic mice engineered to express optogenetic tools in a cell type-specific manner offer a powerful approach for examining the role of particular cells in discrete circuits in a defined and reproducible way. Not surprisingly then, recent years have seen substantial efforts directed towards generating transgenic mouse lines that express functionally relevant levels of optogenetic tools. In this chapter, we review the state of these efforts and consider aspects of the current technology that would benefit from additional improvement.
transgenic mice; genetic manipulation; cell type; Cre; channelrhodopsin; halorhodopsin; archaerhodopsin; calcium indicator; voltage sensor
Fluorescent calcium indicator proteins, such as GCaMP3, allow imaging of activity in genetically defined neuronal populations. GCaMP3 can be expressed using various gene delivery methods, such as viral infection or electroporation. However, these methods are invasive and provide inhomogeneous and non-stationary expression. Here we developed a genetic reporter mouse, Ai38, which expresses GCaMP3 in a Cre-dependent manner from the ROSA26 locus, driven by a strong CAG promoter. Crossing Ai38 with appropriate Cre mice produced robust GCaMP3 expression in defined cell populations in the retina, cortex and cerebellum. In the primary visual cortex, visually-evoked GCaMP3 signals showed normal orientation and direction selectivity. GCaMP3 signals were rapid, compared to virally expressed GCaMP3 and synthetic calcium indicators. In the retina, Ai38 allowed imaging spontaneous calcium waves in starburst amacrine cells during development, and light-evoked responses in ganglion cells in adult tissue. Our results show that the Ai38 reporter mouse provides a flexible method for targeted expression of GCaMP3.
Protein calcium sensor; GECI; reporter mouse; recombinase; orientation selectivity; microcircuitry; OGB-1
The Cre/lox system is widely used in mice to achieve cell-type-specific gene expression. However, a strong and universal responding system to express genes under Cre control is still lacking. We have generated a set of Cre reporter mice with strong, ubiquitous expression of fluorescent proteins of different spectra. The robust native fluorescence of these reporters enables direct visualization of fine dendritic structures and axonal projections of the labeled neurons, which is useful in mapping neuronal circuitry, imaging and tracking specific cell populations in vivo. Using these reporters and a high-throughput in situ hybridization platform, we are systematically profiling Cre-directed gene expression throughout the mouse brain in a number of Cre-driver lines, including novel Cre lines targeting different cell types in the cortex. Our expression data are displayed in a public online database to help researchers assess the utility of various Cre-driver lines for cell-type-specific genetic manipulation.
The putative excitatory and inhibitory cell classes within the mouse primary visual cortex V1 have different functional properties as studied using recording microelectrode. Excitatory neurons show high selectivity for the orientation angle of moving gratings while the putative inhibitory neurons show poor selectivity. However, the study of selectivity of the genetically identified interneurons and their subtypes remain controversial. Here we use novel Cre-driver and reporter mice to identify genetic subpopulations in vivo for two-photon calcium dye imaging: Wfs1(+)/Gad1(−) mice that labels layer 2/3 excitatory cell population and Pvalb(+)/Gad1(+) mice that labels a genetic subpopulation of inhibitory neurons. The cells in both mice were identically labeled with a tdTomato protein, visible in vivo, using a Cre-reporter line. We found that the Wfs1(+) cells exhibited visual tuning properties comparable to the excitatory population, i.e., high selectivity and tuning to the angle, direction, and spatial frequency of oriented moving gratings. The functional tuning of Pvalb(+) neurons was consistent with previously reported narrow-spiking interneurons in microelectrode studies, exhibiting poorer selectivity than the excitatory neurons. This study demonstrates the utility of Cre-transgenic mouse technology in selective targeting of subpopulations of neurons and makes them amenable to structural, functional, and connectivity studies.
orientation preference; GABAergic; somatic inhibition; microcircuitry; cre; reporter mouse; cell type
Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.
We describe a technology for the creation of inducible and reversible gene inactivation in mice. It combines two genetically modified mouse lines: a knock-out line with a tetracycline transactivator replacing the endogenous target gene, and a line in which a tetracycline-inducible cDNA of the target gene has been inserted into a specific genomic locus. A critical component of this system is the unique chromosomal loci we have identified and engineered that offer a platform for easy insertion of any gene of interest for tightly controlled expression. Because of its simple binary nature, allowing independent modification of each of the two components and possibility of use in a high-throughput mode, we believe that our system will be useful for multiple applications, such as introducing mutant or humanized form of the target gene as well as functional manipulating tools. We have applied this technology to the Apolipoprotein E (ApoE) gene and have demonstrated that: a) the expression of ApoE is strictly dependent on the presence of doxycycline, a tetracycline group antibiotic, in the mouse diet, b) in the absence of doxycycline (ApoE repressed) atherosclerotic plaques are formed, confirming the importance of ApoE in the process, and c) upon re-induction of ApoE in the animals with doxicyclin, atherosclerosis regressed.