Patent Grant
8932562
Anxiety is a sustained state of heightened apprehension in the absence of immediate threat, which in disease states becomes severely debilitating. Anxiety disorders represent the most common of the psychiatric diseases (with 28% lifetime prevalence), and have been linked to the etiology of major depression and substance abuse. While the amygdala, a brain region important for emotional processing, has long been hypothesized to play a role in anxiety, the neural mechanisms which control and mediate anxiety have yet to be identified. Despite the high prevalence and severity of anxiety disorders, the corresponding neural circuit substrates are poorly understood, impeding the development of safe and effective treatments. Available treatments tend to be inconsistently effective or, in the case of benzodiazepines, addictive and linked to significant side effects including sedation and respiratory suppression that can cause cognitive impairment and death. A deeper understanding of anxiety control mechanisms in the mammalian brain is necessary to develop more efficient treatments that have fewer side-effects. Of particular interest and novelty would be the possibility of recruiting native pathways for anxiolysis.
Provided herein is an animal comprising a light-responsive opsin expressed in glutamatergic pyramidal neurons of the basolateral amygdala (BLA), wherein the selective illumination of the opsin in the BLA-CeL induces anxiety or alleviates anxiety of the animal.
Provided herein is an animal comprising a light-responsive opsin expressed in glutamatergic pyramidal neurons of the BLA, wherein the opsin is an opsin which induces hyperpolarization by light, and wherein the selective illumination of the opsin in the BLA-CeL induces anxiety of the animal. In some embodiments, the opsin is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1, 2, or 3. In some embodiments, the animal further comprises a second light-responsive opsin expressed in glutamatergic pyramidal neurons of the BLA, wherein the second opsin is an opsin that induces depolarization by light, and wherein the selective illumination of the second opsin in the BLA-CeL reduces anxiety of the animal. In some embodiments, the second opsin is ChR2, VChR1, or DChR. In some embodiments, the second opsin is a C1V1 chimeric protein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the second opsin comprises the amino acid sequence of SEQ ID NO:6 or 7.
Provided herein is an animal comprising a light-responsive opsin expressed in the glutamatergic pyramidal neurons of the BLA, wherein the opsin is an opsin that induces depolarization by light, and wherein the selective illumination of the opsin in the BLA-CeL reduces anxiety of the animal. In some embodiments, the opsin is ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimeric protein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7.
Also provided herein is a vector for delivering a nucleic acid to glutamatergic pyramidal neurons of the BLA in an individual, wherein the vector comprises the nucleic acid encoding a light-responsive opsin and the nucleic acid is operably linked to a promoter that controls the specific expression of the opsin in the glutamatergic pyramidal neurons. In some embodiments, the promoter is a CaMKIIα promoter. In some embodiments, the vector is an AAV vector. In some embodiments, the opsin is an opsin that induces depolarization by light, and wherein selective illumination of the opsin in the BLA-CeL alleviates anxiety. In some embodiments, the opsin that induces depolarization by light is ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimeric protein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7. In some embodiments, the opsin is an opsin that induces hyperpolarization by light, and wherein selective illumination of the opsin in the BLA-CeL and induces anxiety. In some embodiments, the opsin that induces hyperpolarization by light is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1, 2, or 3. In some embodiments, the individual is a mouse or a rat. In some embodiments, the individual is a human.
Also provided here is a method of delivering a nucleic acid to glutamatergic pyramidal neurons of the BLA in an individual, comprising administering to the individual an effective amount of a vector comprising a nucleic acid encoding a light-responsive opsin and the nucleic acid is operably linked to a promoter that controls the specific expression of the opsin in the glutamatergic pyramidal neurons. In some embodiments, the promoter is a CaMKIIα promoter. In some embodiments, the vector is an AAV vector. In some embodiments, the opsin is an opsin that induces depolarization by light, and wherein selective illumination of the opsin in the BLA-CeL alleviates anxiety. In some embodiments, the opsin that induces depolarization by light is ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimeric protein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7. In some embodiments, the opsin is an opsin that induces hyperpolarization by light, and wherein selective illumination of the opsin in the BLA-CeL and induces anxiety. In some embodiments, the opsin that induces hyperpolarization by light is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1, 2, or 3. In some embodiments, the individual is a mouse or a rat. In some embodiments, the individual is a human.
Also provided herein is a coronal brain tissue slice comprising BLA, CeL, and CeM, wherein a light-responsive opsin is expressed in the glutamatergic pyramidal neurons of the BLA. In some embodiments, the opsin is an opsin that induces depolarization by light. In some embodiments, the opsin that induces depolarization by light is ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimeric protein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7. In some embodiments, the opsin is an opsin that induces hyperpolarization by light. In some embodiments, the opsin that induces hyperpolarization by light is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1, 2, or 3. In some embodiments, the tissue is a mouse or a rat tissue.
Also provided herein is a method for screening for a compound that alleviates anxiety, comprising (a) administering a compound to an animal having anxiety induced by selectively illumination of an opsin expressed in the glutamatergic pyramidal neurons of the BLA, wherein the animal comprises a light-responsive opsin expressed in the glutamatergic pyramidal neurons of the BLA, wherein the opsin is an opsin that induces hyperpolarization by light; and (b) determining the anxiety level of the animal, wherein a reduction of the anxiety level indicates that the compound may be effective in treating anxiety. In some embodiments, the opsin is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1, 2, or 3.
Also provided herein is a method for alleviating anxiety in an individual, comprising: (a) administering to the individual an effective amount of a vector comprising a nucleic acid encoding a light-responsive opsin and the nucleic acid is operably linked to a promoter that controls the specific expression of the opsin in the glutamatergic pyramidal neurons of the BLA, wherein the opsin is expressed in the glutamatergic pyramidal neurons of the BLA, wherein the opsin is an opsin that induces depolarization by light; and (b) selectively illuminating the opsin in the glutamatergic pyramidal neurons in the BLA-CeL to alleviate anxiety. In some embodiments, the promoter is a CaMKIIα promoter. In some embodiments, the vector is an AAV vector. In some embodiments, the opsin is ChR2, VChR1, or DChR. In some embodiments, the opsin is a C1V1 chimeric protein comprising the amino acid sequence of SEQ ID NO:8, 9, 10, or 11. In some embodiments, the opsin comprises the amino acid sequence of SEQ ID NO:6 or 7.
Also provided herein is a method for inducing anxiety in an individual, comprising: (a) administering to the individual an effective amount of a vector comprising a nucleic acid encoding an opsin and the nucleic acid is operably linked to a promoter that controls the specific expression of the opsin in the glutamatergic pyramidal neurons of the BLA, wherein the opsin is expressed in the glutamatergic pyramidal neurons, wherein the opsin is an opsin that induces hyperpolarization by light; and (b) selectively illuminating the opsin in the glutamatergic pyramidal neurons in the BLA-CeL to induce anxiety. In some embodiments, the promoter is a CaMKIIα promoter. In some embodiments, the vector is an AAV vector. In some embodiments, the opsin is NpHR, BR, AR, or GtR3. In some embodiments, the NpHR comprises the amino acid sequence of SEQ ID NO:1, 2, or 3.
It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of skill in the art.
Various example embodiments may be more completely understood in consideration of the following description and the accompanying drawings, in which:
The present disclosure relates to control over nervous system disorders, such as disorders associated with anxiety and anxiety symptoms, as described herein. While the present disclosure is not necessarily limited in these contexts, various aspects of the invention may be appreciated through a discussion of examples using these and other contexts.
Various embodiments of the present disclosure relate to an optogenetic system or method that correlates temporal control over a neural circuit with measurable metrics. For instance, various metrics or symptoms might be associated with a neurological disorder exhibiting various symptoms of anxiety. The optogenetic system targets a neural circuit within a patient for selective control thereof. The optogenetic system involves monitoring the patient for the metrics or symptoms associated with the neurological disorder. In this manner, the optogenetic system can provide detailed information about the neural circuit, its function and/or the neurological disorder.
Consistent with the embodiments discussed herein, particular embodiments relate to studying and probing disorders. Other embodiments relate to the identification and/or study of phenotypes and endophenotypes. Still other embodiments relate to the identification of treatment targets.
Aspects of the present disclosure are directed to using an artificially-induced anxiety state for the study of anxiety in otherwise healthy animals. This can be particularly useful for monitoring symptoms and aspects that are poorly understood and otherwise difficult to accurately model in living animals. For instance, it can be difficult to test and/or study anxiety states due to the lack of available animals exhibiting the anxiety state. Moreover, certain embodiments allow for reversible anxiety states, which can be particularly useful in establishing baseline/control points for testing and/or for testing the effects of a treatment on the same animal when exhibiting the anxiety state and when not exhibiting the anxiety state. The reversible anxiety states of certain embodiments can also allow for a reset to baseline between testing the effects of different treatments on the same animal.
Certain aspects of the present disclosure are directed to a method related to control over anxiety and/or anxiety symptoms in a living animal. In certain more specific embodiments, the monitoring of the symptoms also includes assessing the efficacy of the stimulus in mitigating the symptoms of anxiety. Various other methods and applications exist, some of which are discussed in more detail herein.
Light-responsive opsins that may be used in the present invention includes opsins that induce hyperpolarization in neurons by light and opsins that induce depolarization in neurons by light. Examples of opsins are shown in Tables 1 and 2 below.
Table 1 shows identified opsins for inhibition of cellular activity across the visible spectrum:
As used herein, a light-responsive opsin (such as NpHR, BR, AR, GtR3, Mac, ChR2, VChR1, DChR, and ChETA) includes naturally occurring protein and functional variants, fragments, fusion proteins comprising the fragments or the full length protein. For example, the signal peptide may be deleted. A variant may have an amino acid sequence at least about any of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the naturally occurring protein sequence. A functional variant may have the same or similar hyperpolarization function or depolarization function as the naturally occurring protein.
In some embodiments, the NpHR is eNpHR3.0 or eNpHR3.1 (See www.stanford.edu/group/dlab/optogenetics/sequence_info.html). In some embodiments, the light-responsive opsin is a C1V1 chimeric protein or a C1V1-E162 (SEQ ID NO:10), C1V1-E122 (SEQ ID NO:9), or C1V1-E122/E162 (SEQ ID NO:11) mutant chimeric protein (See, Yizhar et al, Nature, 2011, 477(7363):171-78 and www.stanford.edu/group/dlab/optogenetics/sequence_info.html). In some embodiments, the light-responsive opsin is a SFO (SEQ ID NO:6) or SSFO (SEQ ID NO:7) (See, Yizhar et al, Nature, 2011, 477(7363):171-78; Berndt et al., Nat. Neurosci., 12(2):229-34 and www.stanford.edu/group/dlab/optogenetics/sequence_info.html).
In some embodiments, the light-activated protein is a NpHR opsin comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to the sequence shown in SEQ. ID NO:1. In some embodiments, the NpHR opsin further comprises an endoplasmic reticulum (ER) export signal and/or a membrane trafficking signal. For example, the NpHR opsin comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1 and an endoplasmic reticulum (ER) export signal. In some embodiments, the amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1 is linked to the ER export signal through a linker. In some embodiments, the ER export signal comprises the amino acid sequence FXYENE, where X can be any amino acid. In another embodiment, the ER export signal comprises the amino acid sequence VXXSL, where X can be any amino acid. In some embodiments, the ER export signal comprises the amino acid sequence FCYENEV. In some embodiments, the NpHR opsin comprises an amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, an ER export signal, and a membrane trafficking signal. In other embodiments, the NpHR opsin comprises, from the N-terminus to the C-terminus, the amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, the ER export signal, and the membrane trafficking signal. In other embodiments, the NpHR opsin comprises, from the N-terminus to the C-terminus, the amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1, the membrane trafficking signal, and the ER export signal. In some embodiments, the membrane trafficking signal is derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the membrane trafficking signal comprises the amino acid sequence K S R I T S E G E Y I P L D Q I D I N V. In some embodiments, the membrane trafficking signal is linked to the amino acid sequence at least 95% identical to the sequence shown in SEQ ID NO:1 by a linker. In some embodiments, the membrane trafficking signal is linked to the ER export signal through a linker. The linker may comprise any of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 400, or 500 amino acids in length. The linker may further comprise a fluorescent protein, for example, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein. In some embodiments, the light-activated opsin further comprises an N-terminal signal peptide. In some embodiments, the light-activated opsin comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the light-activated protein comprises the amino acid sequence of SEQ ID NO:3.
In some embodiments, the light-activated opsin is a chimeric protein derived from VChR1 from Volvox carteri and ChR1 from Chlamydomonas reinhardti. In some embodiments, the chimeric protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the corresponding first and second transmembrane helices of ChR1. In other embodiments, the chimeric protein comprises the amino acid sequence of VChR1 having the first and second transmembrane helices replaced by the corresponding first and second transmembrane helices of ChR1 and further comprises at least a portion of the intracellular loop domain located between the second and third transmembrane helices replaced by the corresponding portion from ChR1. In some embodiments, the entire intracellular loop domain between the second and third transmembrane helices of the chimeric light-activated protein can be replaced with the corresponding intracellular loop domain from ChR1. In some embodiments, the light-activated chimeric protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:8 without the signal peptide sequence. In some embodiments, the light-activated chimeric protein comprises an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence shown in SEQ ID NO:8. C1V1 chimeric light-activated opsins that may have specific amino acid substitutions at key positions throughout the retinal binding pocket of the VChR1 portion of the chimeric polypeptide. In some embodiments, the C1V1 protein has a mutation at amino acid residue E122 of SEQ ID NO:8. In some embodiments, the C1V1 protein has a mutation at amino acid residue E162 of SEQ ID NO:8. In other embodiments, the C1V1 protein has a mutation at both amino acid residues E162 and E122 of SEQ ID NO:8. In some embodiments, each of the disclosed mutant C1V1 chimeric proteins can have specific properties and characteristics for use in depolarizing the membrane of an animal cell in response to light.
As used herein, a vector comprises a nucleic acid encoding a light-responsive opsin described herein and the nucleic acid is operably linked to a promoter that controls the specific expression of the opsin in the glutamatergic pyramidal neurons. Any vectors that are useful for delivering a nucleic acid to glutamatergic pyramidal neurons may be used. Vectors include viral vectors, such as AAV vectors, retroviral vectors, adenoviral vectors, HSV vectors, and lentiviral vectors. Examples of AAV vectors are AAV 1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV 11, AAV12, AAV13, AAV14, AAV15, and AAV16. A CaMKIIα promoter and any other promoters that can control the expression of the opsin in the glutamatergic pyramidal neurons may be used.
An “individual” is a mammal, such as a human. Mammals also include, but are not limited to, farm animals, sport animals, pets (such as cats, dogs, horses), primates, mice and rats. An “animal” is a non-human mammal.
As used herein, “treatment” or “treating” or “alleviation” is an approach for obtaining beneficial or desired results including and preferably clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: showing observable and/or measurable reduction in one or more signs of the disease (such as anxiety), decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or delaying the progression of the disease.
As used herein, an “effective dosage” or “effective amount” of a drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, and/or delaying the progression of the disease. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, pharmaceutical composition, or another treatment. Thus, an “effective dosage” may be considered in the context of administering one or more therapeutic agents or treatments, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents or treatments, a desirable result may be or is achieved.
The above overview is not intended to describe each illustrated embodiment or every implementation of the present disclosure.
Detailed Description and Example Experimental Embodiments
The present disclosure is believed to be useful for controlling anxiety states and/or symptoms of anxiety. Specific applications of the present invention relate to optogenetic systems or methods that correlate temporal, spatio and/or cell-type control over a neural circuit associated with anxiety states and/or symptoms thereof. As many aspects of the example embodiments disclosed herein relate to and significantly build on previous developments in this field, the following discussion summarizes such previous developments to provide a solid understanding of the foundation and underlying teachings from which implementation details and modifications might be drawn, including those found in the Examples. It is in this context that the following discussion is provided and with the teachings in the references incorporated herein by reference. While the present invention is not necessarily limited to such applications, various aspects of the invention may be appreciated through a discussion of various examples using this context.
Anxiety refers to a sustained state of heightened apprehension in the absence of an immediate threat, which in disease states becomes severely debilitating. Embodiments of the present disclosure are directed toward the use of one or more of cell type-specific optogenetic tools with two-photon microscopy, electrophysiology, and anxiety assays to study and develop treatments relating to neural circuits underlying anxiety-related behaviors.
Aspects of the present disclosure are related to the optogenetic targeting of specific projections of the brain, rather than cell types, in the study of neural circuit function relevant to psychiatric disease.
Consistent with particular embodiments of the present disclosure, temporally-precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA) are used to produce a reversible anxiolytic effect. The optogenetic stimulation can be implemented by viral transduction of BLA with a light-responsive opsin, such as ChR2, followed by restricted illumination in downstream CeA.
Consistent with other embodiments of the present disclosure, optogenetic inhibition of the basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA) are used to increase anxiety-related behaviors. The optogenetic stimulation can be implemented by viral transduction of BLA with a light-responsive opsin, such as eNpHR3.0, followed by restricted illumination in downstream CeA.
Embodiments of the present disclosure are directed towards the specific targeting of neural cell populations, as anxiety-based effects were not observed with direct optogenetic control of BLA somata. For instance, targeting of specific BLA-CeA projections as circuit elements have been experimentally shown to be sufficient for endogenous anxiety control in the mammalian brain.
Consistent with embodiments of the present disclosure, the targeting of the specific BLA-CeA projections as circuit elements is based upon a number of factors discussed in more detail hereafter. The amygdala is composed of functionally and morphologically heterogeneous subnuclei with complex interconnectivity. A primary subdivision of the amygdala is the basolateral amygdala complex (BLA), which encompasses the lateral (LA), basolateral (BL) and basomedial (BM) amygdala nuclei (˜90% of BLA neurons are glutamatergic). In contrast, the central nucleus of the amygdala (CeA), which is composed of the centrolateral (CeL) and centromedial (CeM) nuclei, is predominantly (˜95%) comprised of GABAergic medium spiny neurons. The BLA is ensheathed in dense clusters of GABAergic intercalated cells (ITCs), which are functionally distinct from both local interneurons and the medium spiny neurons of the CeA. The primary output nucleus of the amygdala is the CeM, which, when chemically or electrically excited, is believed to mediate autonomic and behavioral responses that are associated with fear and anxiety via projections to the brainstem. While the CeM is not directly controlled by the primary amygdala site of converging environmental and cognitive information (LA), LA and BLA neurons excite GABAergic CeL neurons, which can provide feed-forward inhibition onto CeM “output” neurons and reduce amygdala output. The BLA-CeL-CeM is a less-characterized pathway suggested to be involved not in fear extinction but in conditioned inhibition. The suppression of fear expression, possibly due to explicit unpairing of the tone and shock, suggested to be related to the potentiation of BLA-CeL synapses.
BLA cells have promiscuous projections throughout the brain, including to the bed nucleus of the stria terminalis (BNST), nucleus accumbens, hippocampus and cortex. Aspects of the present disclosure relate to methods for selective control of BLA terminals in the CeL, without little or no direct affect/control of other BLA projections. Preferential targeting of BLA-CeL synapses can be facilitated by restricting opsin gene expression to BLA glutamatergic projection neurons and by restricting light delivery to the CeA.
For instance, control of BLA glutamatergic projection neurons can be achieved with an adeno-associated virus (AAV5) vector carrying light-activated optogenetic control genes under the control of a CaMKIIα promoter. Within the BLA, CaMKIIα is only expressed in glutamatergic pyramidal neurons, not in local interneurons or intercalated cells.
Embodiments of the present disclosure are directed toward the above realization being applied to various ones of the anatomical, functional, structural, and circuit targets identified herein. For instance, the circuit targets can be studied to develop treatments for the psychiatric disease of anxiety. These treatments can include, as non-limiting examples, pharmacological, electrical, magnetic, surgical and optogenetic, or other treatment means.
Various embodiments of the present disclosure relate to the use of the identified model for screening new treatments for anxiety. For instance, anxiety can be artificially induced or repressed using the methods discussed herein, while pharmacological, electrical, magnetic, surgical, or optogenetic treatments are then applied and assessed. In other embodiments of the present disclosure, the model can be used to develop an in vitro approximation or simulation of the identified circuit, which can then be used in the screening of devices, reagents, tools, technologies, methods and approaches and for studying and probing anxiety and related disorders. This study can be directed towards, but not necessarily limited to, identifying phenotypes, endophenotypes, and treatment targets.
Embodiments of the present disclosure are directed toward modeling the BLA-CeL pathway as an endogenous neural substrate for bidirectionally modulating the unconditioned expression of anxiety. Certain embodiments are directed toward other downstream circuits, such as CeA projections to the BNST, for their role in the expression of anxiety or anxiety-related behaviors. For instance, it is believed that corticotropin releasing hormone (CRH) networks in the BNST may be critically involved in modulating anxiety-related behaviors, as the CeL is a primary source of CRH for the BNST. Other neurotransmitters and neuromodulators may modulate or gate effects on distributed neural circuits, including serotonin, dopamine, acetylcholine, glycine, GABA and CRH. Still other embodiments are directed toward control of the neural circuitry converging to and diverging from this pathway, as parallel or downstream circuits of the BLA-CeL synapse are believed to contribute to the modulation or expression of anxiety phenotypes. Moreover, upstream of the amygdala, this microcircuit is well-positioned to be recruited by top-down cortical control from regions important for processing fear and anxiety, including the prelimbic, infralimbic and insular cortices that provide robust innervation to the BLA and CeL.
Experimental results based upon the BLA anatomy suggest that the populations of BLA neurons projecting to CeL and CeM neurons are largely non-overlapping. In natural states, the CeL-projecting BLA neurons may excite CeM-projecting BLA neurons in a microcircuit homeostatic mechanism, which can then be used to study underlying anxiety disorders when there are synaptic changes that skew the balance of the circuit to allow uninhibited CeM activation.
The embodiments and specific applications discussed herein (including the Examples) may be implemented in connection with one or more of the above-described aspects, embodiments and implementations, as well as with those shown in the figures and described below. Reference may be made to the following Example, which is fully incorporated herein by reference. For further details on light-responsive molecules and/or opsins, including methodology, devices and substances, reference may also be made to the following background publications: U.S. Patent Publication No. 2010/0190229, entitled “System for Optical Stimulation of Target Cells” to Zhang et al.; U.S. Patent Publication No. 2010/0145418, also entitled “System for Optical Stimulation of Target Cells” to Zhang et al.; U.S. Patent Publication No. 2007/0261127, entitled “System for Optical Stimulation of Target Cells” to Boyden et al.; and PCT WO 2011/116238, Entitled “Light Sensitive Ion Passing Molecules”. These applications form part of the patent document and are fully incorporated herein by reference. Consistent with these publications, numerous opsins can be used in mammalian cells in vivo and in vitro to provide optical stimulation and control of target cells. For example, when ChR2 is introduced into an electrically-excitable cell, such as a neuron, light activation of the ChR2 channel rhodopsin can result in excitation and/or firing of the cell. In instances when NpHR is introduced into an electrically-excitable cell, such as a neuron, light activation of the NpHR opsin can result in inhibition of firing of the cell. These and other aspects of the disclosures of the above-referenced patent applications may be useful in implementing various aspects of the present disclosure.
While the present disclosure is amenable to various modifications and alternative forms, specifics thereof have been shown by way of example in the drawings and will be described in further detail. It should be understood that the intention is not to limit the disclosure to the particular embodiments and/or applications described. On the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
Introduction
Anxiety is a sustained state of heightened apprehension in the absence of immediate threat, which in disease states becomes severely debilitating'. Anxiety disorders represent the most common of the psychiatric diseases (with 28% lifetime prevalence)2, and have been linked to the etiology of major depression and substance abuse3-5. While the amygdala, a brain region important for emotional processing9-17, has long been hypothesized to play a role in anxiety18-23, the neural mechanisms which control and mediate anxiety have yet to be identified. Here, we combine cell type-specific optogenetic tools with two-photon microscopy, electrophysiology, and anxiety assays in freely-moving mice to identify neural circuits underlying anxiety-related behaviors. Capitalizing on the unique capability of optogenetics24-26 to control not only cell types, but also specific connections between cells, we observed that temporally-precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA), resolved by viral transduction of BLA with ChR2 followed by restricted illumination in downstream CeA, exerted a profound, immediate, and reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same defined projection with eNpHR3.025 potently, swiftly, and reversibly increased anxiety-related behaviors. Importantly, these effects were not observed with direct optogenetic control of BLA somata themselves. Together, these results implicate specific BLA-CeA projections as circuit elements both necessary and sufficient for endogenous anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting specific projections, rather than cell types, in the study of neural circuit function relevant to psychiatric disease.
Despite the high prevalence and severity' of anxiety disorders, the corresponding neural circuit substrates are poorly understood, impeding the development of safe and effective treatments. Available treatments tend to be inconsistently effective or, in the case of benzodiazepines, addictive and linked to significant side effects including sedation and respiratory suppression that can cause cognitive impairment and death27,28. A deeper understanding of anxiety control mechanisms in the mammalian brain29,30 is necessary to develop more efficient treatments that have fewer side-effects. Of particular interest and novelty would be the possibility of recruiting native pathways for anxiolysis.
The amygdala is critically involved in processing associations between neutral stimuli and positive or negative outcomes, and has also been implicated in processing unconditioned emotional states. While the amygdala microcircuit has been functionally dissected in the context of fear conditioning, amygdalar involvement has been implicated in a multitude of other functions and emotional states, including unconditioned anxiety. The amygdala is composed of functionally and morphologically heterogeneous subnuclei with complex interconnectivity. A primary subdivision of the amygdala is the basolateral amygdala complex (BLA), which encompasses the lateral (LA), basolateral (BL) and basomedial (BM) amygdala nuclei (˜90% of BLA neurons are glutamatergic)33,34. In contrast, the central nucleus of the amygdala (CeA), which is composed of the centrolateral (CeL) and centromedial (CeM) nuclei, is predominantly (˜95%) comprised of GABAergic medium spiny neurons35. The BLA is ensheathed in dense clusters of GABAergic intercalated cells (ITCs), which are functionally distinct from both local interneurons and the medium spiny neurons of the CeA36,37. The primary output nucleus of the amygdala is the CeM,32,35, 38-40 which when chemically or electrically excited mediates autonomic and behavioral responses associated with fear and anxiety via projections to the brainstem6, 12, 32, 35. While the CeM is not directly controlled by the primary amygdala site of converging environmental and cognitive information (LA)12, 38, 41, LA and BLA neurons excite GABAergic CeL neurons42 which can provide feed-forward inhibition onto CeM40, 46 “output” neurons and reduce amygdala output. The BLA-CeL-CeM is a less-characterized pathway suggested to be involved not in fear extinction but in conditioned inhibition, the suppression of fear expression due to explicit unpairing of the tone and shock, due to the potentiation of BLA-CeL synapses47. Although fear is characterized to be a phasic state triggered by an external cue, while anxiety is a sustained state that may occur in the absence of an external trigger, we wondered if circuits modulating conditioned inhibition of fear might also be involved in modulating unconditioned inhibition of anxiety.
Materials and Methods
Subjects: Male C57BL/6 mice, aged 4-6 weeks at the start of experimental procedures, were maintained with a reverse 12-hr light/dark cycle and given food and water ad libitum. Animals shown in
Optical Intensity Measurements: Light transmission measurements were conducted with blocks of brain tissue from acutely sacrificed mice. The tissue was then placed over the photodetector of a power meter (ThorLabs, Newton, N.J.) to measure the light power of the laser penetrated the tissue. The tip of a 300 um diameter optical fiber was coupled to a 473 nm blue laser (OEM Laser Systems, East Lansing, Mich.). To characterize the light transmission to the opposite side of the bevel, the photodetector of the power meter was placed parallel to the beveled cannula. For visualization of the light cone, we used Fluorescein isothiocyanate-dextran (FD150s; Sigma, Saint Louis, Mo.) at approximately 5 mg/ml placed in a cuvette with the optical fibers either with or without beveled cannula shielding aimed perpendicularly over the fluorescein solution. Power density at specific depths were calculated considering both fractional decrease in intensity due to the conical output of light from the optical fiber and the loss of light due to scattering in tissue (Aravanis et al., J Neural Eng, 4:S143-156, 2007) (Gradinaru et al., J Neurosci, 27:14231-14238, 2007). The half-angle of divergence θdiv for a multimode optical fiber, which determines the angular spread of the output light, is
where ntis is the index of refraction of gray matter (1.36, Vo-Dinh T 2003, Biomedical Photonics Handbook (Boca Raton, Fla.: CRC Press)) and NAfib(0.37) is the numerical aperture of the optical fiber. The fractional change in intensity due to the conical spread of the light with distance (z) from the fiber end was calculated using trigonometry
and r is the radius of the optical fiber (100 um).
The fractional transmission of light after loss due to scattering was modeled as a hyperbolic function using empirical measurements and the Kubelka-Munk model1, 2, and the combined product of the power density at the tip of the fiber and the fractional changes due to the conical spread and light scattering, produces the value of the power density at a specific depth below the fiber.
Virus construction and packaging: The recombinant AAV vectors were serotyped with AAV5 coat proteins and packaged by the viral vector core at the University of North Carolina. Viral titers were 2×10e12 particles/mL, 3×10e12 particles/mL, 4×10e12 particles/mL respectively for AAV-CaMKIIα-hChR2(H134R)— EYFP, AAV-CaMKIIα-EYFP, and AAV-CaMKIIα-eNpHR 3.0-EYFP. The pAAV-CaMKIIα-eNpHR3.0-EYFP plasmid was constructed by cloning CaMKIIα-eNpHR3.0-EYFP into an AAV backbone using MluI and EcoRI restriction sites. Similarly, The pAAV-CaMKIIα-EYFP plasmid was constructed by cloning CaMKIIα-EYFP into an AAV backbone using MluI and EcoRI restriction sites. The maps are available online at www.optogenetics.org, which are incorporated herein by reference.
Stereotactic injection and optical fiber placement: All surgeries were performed under aseptic conditions under stereotaxic guidance. Mice were anaesthetized using 1.5-3.0% isoflourane. All coordinates are relative to bregma in mm3. In all experiments, both in vivo and in vitro, virus was delivered to the BLA only, and any viral expression in the CeA rendered exclusion from all experiments. Cannula guides were beveled to form a 45-55 degree angle for the restriction of the illumination to the CeA. The short side of the beveled cannula guide was placed antero-medially, the long side of the beveled cannula shielded the posterior-lateral portion of the light cone, facing the opposite direction of the viral injection needle. To preferentially target BLA-CeL synapses, we restricted opsin gene expression to BLA glutamatergic projection neurons and restricted light delivery to the CeA. Control of BLA glutamatergic projection neurons was achieved using an adeno-associated virus (AAV5) vector carrying light-activated optogenetic control genes under the control of a CaMKIIα promoter. Within the BLA, CaMKIIα is only expressed in glutamatergic pyramidal neurons, not in local interneurons4. Mice in the ChR2 Terminals and EYFP Terminals groups received unilateral implantations of beveled cannulae for the optical fiber (counter-balanced for hemisphere), while mice in the eNpHR 3.0 or respective EYFP group received bilateral implantations of the beveled cannulae over the CeA (−1.06 mm anteroposterior (AP); ±2.25 mm mediolateral (ML); and −4.4 mm dorsoventral (DV); PlasticsOne, Roanoke, Va.)3. Mice in the ChR2 Cell Bodies groups received unilateral implantation of a Doric patchcord chronically implantable fiber (NA=0.22; Doric lenses, Quebec, Canada) over the BLA at (−1.6 mm AP; ±3.1 mm ML; −4.5 mm DV)3. For all mice, 0.5 μl of purified AAV5 was injected unilaterally or bilaterally in the BLA (±3.1 mm AP, 1.6 mm ML, −4.9 mm DV)3 using beveled 33 or 35 gauge metal needle facing postero-lateral side to restrict the viral infusion to the BLA. 10 μl Hamilton microsyringe (nanofil; WPI, Sarasota, Fla.) were used to deliver concentrated AAV solution using a microsyringe pump (UMP3; WPI, Sarasota, Fla.) and its controller (Micro4; WPI, Sarasota, Fla.). Then, 0.5 μl of virus solution was injected at each site at a rate of 0.1 μl per min. After injection completion, the needle was lifted 0.1 mm and stayed for 10 additional minutes and then slowly withdrawn. One layer of adhesive cement (C&B metabond; Parkell, Edgewood, N.Y.) followed by cranioplastic cement (Dental cement; Stoelting, Wood Dale, Ill.) was used to secure the fiber guide system to the skull. After 20 min, the incision was closed using tissue adhesive (Vetbond; Fisher, Pittsburgh, Pa.). The animal was kept on a heating pad until it recovered from anesthesia. A dummy cap (rat: C312G, mouse: C313G) was inserted to keep the cannula guide patent. Behavioral and electrophysiological experiments were conducted 4-6 weeks later to allow for viral expression.
In vivo recordings: Simultaneous optical stimulation of central amygdala (CeA) and electrical recording of basolateral amygdala (BLA) of adult male mice previously (4-6 weeks prior) transduced in BLA with AAV-CaMKIIα-ChR2-eYFP viral construct was carried out as described previously (Gradinaru et al., J Neurosci, 27:14231-14238, 2007). Animals were deeply anesthetized with isoflurane prior to craniotomy and had negative toe pinch. After aligning mouse stereotaxically and surgically removing approximately 3 mm2 skull dorsal to amygdala. Coordinates were adjusted to allow for developmental growth of the skull and brain, as mice received surgery when they were 4-6 weeks old and experiments were performed when the mice were 8-10 weeks old (centered at −1.5 mm AP, ±2.75 mm ML)3, a 1 Mohm 0.005-in extracellular tungsten electrode (A-M systems) was stereotactically inserted into the craniotomized brain region above the BLA (in mm: −1.65 AP, ±3.35 ML, −4.9 DV)3. Separately, a 0.2 N.A. 200 μm core diameter fiber optic cable (Thor Labs) was stereotactically inserted into the brain dorsal to CeA (−1.1 AP, ±2.25 ML, −4.2 DV)3. After acquiring a light evoked response, voltage ramps were used to vary light intensity during stimulation epochs (20 Hz, 5 ms pulse width) 2 s in length. After acquiring optically evoked signal, the exact position of the fiber was recorded, the fiber removed from the brain, inserted into a custom beveled cannula, reinserted to the same position, and the same protocol was repeated. In most trials, the fiber/cannula was then extracted from the brain, the cannula removed, and the bare fiber reinserted to ensure the fidelity of the population of neurons emitting the evoked signal. Recorded signals were bandpass filtered between 300 Hz and 20 kHz, AC amplified either 1000× or 10000×(A-M Systems 1800), and digitized (Molecular Devices Digidata 1322A) before being recorded using Clampex software (Molecular Devices). Clampex software was used for both recording field signals and controlling a 473 nm (OEM Laser Systems) solidstate laser diode source coupled to the optrode. Light power was titrated between <1 mW (−14 mW/mm2) and 28 mW (−396 mW/mm2) from the fiber tip and measured using a standard light power meter (ThorLabs). Electrophysiological recordings were initiated approximately 1 mm dorsal to BLA after lowering isoflurane anesthesia to a constant level of 1%. Optrode was lowered ventrally in ˜0.1 mm steps until localization of optically evoked signal.
Behavioral assays: All animals used for behavior received viral transduction of BLA neurons and the implantation enabling unilateral (for ChR2 groups and controls) or bilateral (for eNpHR3.0 groups and controls) light delivery. For behavior, multimode optical fibers (NA 0.37; 300 μm core, BFL37-300; ThorLabs, Newton, N.J.) were precisely cut to the optimal length for restricting the light to the CeA, which was shorter than the long edge of the beveled cannula, but longer than the shortest edge of the beveled cannula. For optical stimulation, the fiber was connected to a 473 nm or 594 nm laser diode (OEM Laser Systems, East Lansing, Mich.) through an FC/PC adapter. Laser output was controlled using a Master-8 pulse stimulator (A.M.P.I., Jerusalem, Israel) to deliver light trains at 20 Hz, 5 ms pulse-width for 473 nm light, and constant light for 594 nm light experiments. All included animals had the center of the viral injection located in the BLA, though there was sometimes leak to neighboring regions or along the needle tract. Any case in which there was any detectable viral expression in the CeA, the animals were excluded. All statistically significant effects of light were discussed, and undiscussed comparisons did not show detectable differences.
The elevated plus maze was made of plastic and consisted of two light gray open arms (30×5 cm), two black enclosed arms (30×5×30 cm) extending from a central platform (5×5×5 cm) at 90 degrees in the form of a plus. The maze was placed 30 cm above the floor. Mice were individually placed in the center. 1-5 minutes were allowed for recovery from handling before the session was initiated. Video tracking software (BiObserve, Fort Lee, N.J.) was used to track mouse location, velocity and movement of head, body and tail. All measurements displayed were relative to the mouse body. Light stimulation protocols are specified by group. ChR2:BLA-CeA mice and corresponding controls groups (EYFP:BLA-CeA and ChR2:BLA Somata) were singly-housed in a high-stress environment for at least 1 week prior to anxiety assays: unilateral illumination of BLA terminals in the CeA at 7-8 mW (−106 mW/mm2 at the tip of the fiber, ˜6.3 mW/mm2 at CeL and ˜2.4 mW/mm2 at the CeM) of 473 nm light pulse trains (5 ms pulses at 20 Hz). For the ChR2 Cell Bodies group BLA neurons were directly illuminated with a lower light power because illumination with 7-8 mW induced seizure activity, so we unilaterally illuminated BLA neurons at 3-5 mW (˜57 mW/mm2) of 473 nm light pulse trains (5 ms pulses at 20 Hz). For the eNpHR 3.0 and corresponding EYFP group, all mice were group-housed and received bilateral viral injections and bilateral illumination of BLA terminals in the CeA at 4-6 mW (−71 mW/mm2 at the tip of the fiber, ˜4.7 mW/mm2 at the CeL and ˜1.9 mW/mm2 at the CeM) of 594 nm light with constant illumination throughout the 5-min light on epoch. The 15-min session was divided into 3 5-min epochs, the first epoch there was no light stimulation (off), the second epoch light was delivered as specified above (on), and the third epoch there was no light stimulation (off).
The open-field chamber (50×50 cm) and the open field was divided into a central field (center, 23×23 cm) and an outer field (periphery). Individual mice were placed in the periphery of the field and the paths of the animals were recorded by a video camera. The total distance traveled was analyzed by using the same video-tracking software, Viewer2 (BiObserve, Fort Lee, N.J.). The open field assessment was made immediately after the elevated-plus maze test. The open field test consisted of an 18-min session in which there were six 3-min epochs. The epochs alternated between no light and light stimulation periods, beginning with a light off epoch. For all analyses and charts where only “off” and “on” conditions are displayed, the 3 “off” epochs were pooled and the 3 “on” epochs were pooled.
For the glutamate receptor antagonist manipulation, a glutamate antagonist solution consisting of 22.0 mM of NBQX and 38.0 mM of D-APV (Tocris, Ellisville, Mo.) dissolved in saline (0.9% NaCl). 5-15 min before the anxiety assays, 0.3 μl of the glutamate antagonist solution was infused into the CeA via an internal infusion needle, inserted into the same guide cannulae used for light delivery via optical fiber, that was connected to a 10-μl Hamilton syringe (nanofil; WPI, Sarasota, Fla.). The flow rate (0.1 μl per min) was regulated by a syringe pump (Harvard Apparatus, MA). Placements of the viral injection, guide cannula and chronically-implanted fiber were histologically verified as indicated in
Two-photon optogenetic circuit mapping and ex vivo electrophysiological recording: Mice were injected with AAV5-CaMKIIα-ChR2-EYFP at 4 weeks of age, and were sacrificed for acute slice preparation 4-6 weeks to allow for viral expression. Coronal slices containing the BLA and CeA were prepared to examine the functional connectivity between the BLA and the CeA. Two-photon images and electrophysiological recordings were made under the constant perfusion of aCSF, which contained (in mM): 126 NaCl, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 1 MgCl2, 2 CaCl2, and 10 glucose. All recordings were at 32° C. Patch electrodes (4-6 MOhms) were filled (in mM): 10 HEPES, 4 Mg-ATP, 0.5 MgCl2, 0.4 Na3-GTP, 10 NaCl, 140 potassium gluconate, and 80 Alexa-Fluor 594 hydrazide (Molecular Probes, Eugene Oreg.). Whole-cell patch-clamp recordings were performed in BLA, CeL and CeM neurons, and cells were allowed to fill for approximately 30 minutes before imaging on a modified two-photon microscope (Prairie Microscopes, Madison Wis.) where two-photon imaging, whole-cell recording and optogenetic stimulation could be done simultaneously. Series resistance of the pipettes was usually 10-20 MOhms Blue light pulses were elicited using a 473 nm LED at ˜7 mW/mm2 (Thorlabs, Newton N.J.) unless otherwise noted. A Coherent Ti-Saphire laser was used to image both ChR2-YFP (940 nm) and Alexa-Fluor 594 (800 nm). A FF560 dichroic with filters 630/69 and 542/27 (Semrock, Rochester N.Y.) was also used to separate both molecules' emission. All images were taken using a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). In order to isolate fibers projecting to CeL from the BLA and examine responses in the CeM, slices were prepared as described above with the BLA excluded from illumination. Whole-cell recordings were performed in the CeM with illumination from the objective aimed over the CeL. To further ensure activation of terminals from the BLA to CeL was selective, illumination was restricted to a ˜125 μm diameter around the center of the CeL. Here, blue light pulses were elicited using an XCite halogen light source (EXPO, Mississauga, Ontario) with a 470/3 filter at 6.5 mW/mm2 coupled to a shutter (Uniblitz, Rochester N.Y.). For functional mapping, we first recorded from a BLA neuron expressing ChR2 and simultaneously collected electrophysiological recordings and filled the cell with Alexa-Fluor 594 hydrazide dye to allow for two-photon imaging. Two-photon z-stacks were collected at multiple locations along the axon of the filled BLA neuron. We then followed the axon of the BLA neuron projecting to the CeL nucleus and recorded from a CeL neuron in the BLA terminal field. We then simultaneously recorded from a CeL neuron, filled the cell with dye and performed two-photon live imaging before following the CeL neuronal axons to the CeM. We then repeated this procedure in a CeM neuron, but moved the light back to the terminal field in the CeL to mimic the preferential illumination of BLA-CeL synapses with the same stimulation parameters as performed in vivo. Voltage-clamp recordings were made at both −70 mV, to isolate EPSCs, and at 0 mV, to isolate IPSCs. EPSCs were confirmed to be EPSCs via bath application of the glutamate receptor antagonists (n=5), NBQX (22 μM) and AP5 (38 μM), IPSCs were confirmed to be IPSCs via bath application of bicuculline (10 μM; n=2), which abolished them, respectively. We also performed current-clamp recordings when the cell was resting at approximately −70 mV.
For the characterization of optogenetically-driven antidromic stimulation in BLA axon terminals, animals were injected with AAV5-CaMKIIα-ChR2-EYFP at 4 weeks of age, and were sacrificed for acute slice preparation 4-6 weeks to allow for viral expression. Slice preparation was the same as above. To the aCSF we added 0.1 mM picrotoxin, 10 μM CNQX and 25 μM AP5 (Sigma, St. Louis, Mo.). Whole-cell patch-clamp recordings were performed in BLA neurons and were allowed to fill for approximately 30 minutes before two-photon imaging. Series resistance of the pipettes was usually 10-20 MOhms. All images were taken using a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Blue light pulses were elicited using an XCite halogen light source (EXPO, Mississauga, Ontario) with a 470/30 filter at 6.5 mW/mm2 coupled to a shutter (Uniblitz, Rochester N.Y.). Two-photon z-stacks were collected at multiple locations along the axon of the filled BLA neuron. Only neurons whose axons could be visualized for over ˜300 μm diameter towards the CeL nucleus were included for the experiment, and neurons that had processes going in all directions were also excluded. Stimulation on/off axon was accomplished by moving the slice relative to a ˜125 μm diameter blue light spot. In order to calibrate the slice for correct expression, whole-cell patch-clamp was performed on a CeL cell and a ˜125 μm diameter spot blue pulse was used to ensure that synaptic release from the BLA terminals on to the CeL neuron was reliable.
For the dissection of direct and indirect projections to CeM, animals were injected with AAV5-CaMKIIα-ChR2-EYFP at 4 weeks of age, and were sacrificed for acute slice preparation 4-6 weeks to allow for viral expression. Slice preparation was the same as above. Light was delivered through a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Prior to whole cell patch clamping in the CeM nucleus, the location of the CeL nucleus was noted in order to revisit it with the light spot restricted to this region. Whole-cell patch-clamp recordings were performed in CeM neurons. Series resistance of the pipettes was usually 10-20 MOhms. Blue light pulses were elicited using a XCite halogen light source (EXPO, Mississauga, Ontario) with a 470/30 filter at 6.5 mW/mm2 coupled to a shutter (Uniblitz, Rochester N.Y.). During CeM recordings, broad illumination (˜425-450 μm in diameter) of BLA terminals in the CeA and 20 Hz, 5 ms light train for 2 s was applied. Voltage-clamp recordings were made at 70 mV and 0 mV to isolate EPSCs and IPSCs respectively. Current-clamp recordings were also made. Then, illumination was moved to the CeL using a restricted light spot ˜125 μm in diameter. We again performed voltage clamp recordings at −70 mV and 0 mV and used 20 Hz, 5 ms light train for 2 s. For the CeM neuron spiking inhibition experiments, in current-clamp, we applied the minimal current step required to induce spiking (˜60 pA) and simultaneously applied preferential illumination of ChR2-expressing BLA terminals in the CeL with a 20 Hz, 5 ms light train for 2 s (mean over 6 sweeps per cell). For the experiments comparing the broad illumination of the BLA terminal field centered in the CeM to selective illumination of BLA-CeL terminals, these conditions were performed in repeated alternation in the same CeM cells (n=7).
To verify that terminal inhibition did not alter somatic spiking, animals were injected with AAV5-CaMKIIα-eNpHR3.0-EYFP at 4 weeks of age, and were sacrificed for acute slice preparation 4-6 weeks to allow for viral expression. Slice preparation was the same as above. Whole-cell patch-clamp recordings were performed in BLA neurons and were allowed to fill for approximately 30 minutes. Light was delivered through a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Whole-cell patch-clamp recordings were performed on BLA neurons. Series resistance of the pipettes was usually 10-20 MOhms. Yellow light pulses were elicited using a XCite halogen light source (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5 mW/mm2 coupled to a shutter (Uniblitz, Rochester N.Y.). After patching, an unrestricted light spot (−425-450 microns in diameter) was placed over the BLA soma and a 1 s pulse was applied. Cells were excluded if the current recorded was under 600 pA of hyperpolarizing current and the axon did not travel over ˜300 μm towards the CeL nucleus. The light spot was then restricted to ˜125 in diameter. On and off axon voltage clamp recordings were taken with a 1 s pulse of light. For the current clamp recordings, action potentials were generated by applying 250 pA of current to the cell soma through the patch pipette.
To demonstrate that selective illumination of eNpHR3.0-expressing BLA terminals reduced the probability of spontaneous vesicle release, animals were injected with AAV5-CaMKIIα-eNpHR3.0-EYFP at 4 weeks of age, and were sacrificed for acute slice preparation 4-6 weeks to allow for viral expression. Slice preparation was the same as above. Whole-cell patch-clamp recordings were performed in central lateral neurons. Light was delivered through a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Series resistance of the pipettes was usually 10-20 MOhms Yellow light pulses were elicited using a XCite halogen light source (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5 mW/mm2 coupled to a shutter (Uniblitz, Rochester N.Y.). The light spot was restricted to ˜125 μm in diameter. Carbachol was added to the bath at a concentration of 20 μM. After sEPSC activity increased in the CeL neuron, light pulses were applied ranging in times from 5 s to 30 s.
To demonstrate that selective illumination of eNpHR3.0-expressing BLA terminals could reduce the probability of vesicle release evoked by electrical stimulation, animals were injected with AAV5-CaMKIIα-eNpHR3.0-EYFP at 4 weeks of age, and were sacrificed for acute slice preparation 4-6 weeks to allow for viral expression. Slice preparation was the same as above. A bipolar concentric stimulation probe (FHC, Bowdoin Me.) was placed in the BLA. Whole-cell patch-clamp recordings were performed in CeL neurons. Light was delivered through a 40×/0.8 NA LUMPlanFL/IR Objective (Olympus, Center Valley Pa.). Series resistance of the pipettes was usually 10-20 MOhms. Amber light pulses over the central lateral cell were elicited using a XCite halogen light source (EXPO, Mississauga, Ontario) with a 589/24 filter at 6.5 mW/mm2 coupled to a shutter (Uniblitz, Rochester N.Y.). The light spot was restricted to ˜125 μm in diameter. Electrical pulses were delivered for 40 seconds and light was delivered starting at 10 seconds and shut off at 30 seconds in the middle.
For the anatomical tracing experiments, neurons were excluded when the traced axons were observed to be severed and all BLA neurons included in the anatomical assay (
Slice immunohistochemistry: Anesthetized mice were transcardially perfused with ice-cold 4% paraformaldehyde (PFA) in PBS (pH 7.4) 100-110 min after termination of in vivo light stimulation. Brains were fixed overnight in 4% PFA and then equilibrated in 30% sucrose in PBS. 40 μm-thick coronal sections were cut on a freezing microtome and stored in cryoprotectant at 4° C. until processed for immunohistochemistry. Free-floating sections were washed in PBS and then incubated for 30 min in 0.3% Tx100 and 3% normal donkey serum (NDS). Primary antibody incubations were performed overnight at 4° C. in 3% NDS/PBS (rabbit anti-c-fos 1:500, Calbiochem, La Jolla, Calif.; mouse anti-CaMKII 1:500, Abcam, Cambridge, Mass.). Sections were then washed and incubated with secondary antibodies (1:1000) conjugated to Cy3 or Cy5 (Jackson Laboratories, West Grove, Pa.) for 3 hrs at room temperature. Following a 20 min incubation with DAPI (1:50,000) sections were washed and mounted on microscope slides with PVD-DABCO.
Confocal microscopy and analysis: Confocal fluorescence images were acquired on a Leica TCS SP5 scanning laser microscope using a 20×/0.70NA or a 40×/1.25NA oil immersion objective. Serial stack images covering a depth of 10 μm through multiple sections were acquired using equivalent settings. The Volocity image analysis software (Improvision/PerkinElmer, Waltham, Mass.) calculated the number of c-fos positive cells per field by thresholding c-fos immunoreactivity above background levels and using the DAPI staining to delineate nuclei. All imaging and analysis was performed blind to the experimental conditions.
Statistics: For behavioral experiments and the ex vivo electrophysiology data, binary comparisons were tested using nonparametric bootstrapped t-tests (paired or unpaired where appropriate)5, while hypotheses involving more than two group means were tested using linear contrasts (using the “boot” and “lme4” packages in R6, respectively); the latter were formulated as contrasts between coefficients of a linear mixed-effects model (a “two-way repeated-measures ANOVA”) with the fixed effects being the genetic or pharmacological manipulation and the light treatment (on or off). All hypothesis tests were specified a priori. Subjects were modeled as a random effects. For c-fos quantification comparisons, we used a one-way ANOVA followed by Tukey's multiple comparisons test.
Plots of the data clearly show a relationship between observation mean and observation variance (that is, they are heteroskedastic; see for example,
√{square root over (yijk)}=μ+ci+tj+(c:t)ij+bj+eijk (1)
where
Collecting the fixed effects into a 2-way analysis of variance (ANOVA) design matrix X∈Rnxp, dummy coding the random effects in a sparse matrix Z∈Rnxq, and letting {tilde over (y)}=√{square root over (y)} we can express the model in matrix form as
{tilde over (y)}=Xβ+Xb+e (2)
where {tilde over (y)}∈n, b∈
q, and ε∈
n are observations of random variables {tilde over (y)}, β, and ε respectively and our model assumes
β˜N(0,σ2Σ)
ε˜N(0,σ2I),ε⊥β
({tilde over (y)}|β=b)˜N(Xβ+Zb,σ2I)
where N (μ,Σ) denotes the multivariate Gaussian distribution with mean vector μ and variance-covariance matrix Σ, and ⊥ indicates that two variables are independent. To estimate the coefficient vectors β∈Rp, b∈Rq, and the variance parameter σ and sparse (block-diagonal) relative variance-covariance matrix Σ∈Rqxq, we use the lme4 package in R written by Douglas Bates and Martin Maechler, which first finds a linear change of coordinates that “spheres” the random effects and then finds the maximum likelihood estimates for β, σ, and Σ using penalized iteratively reweighted least-squares, exploiting the sparsity of the random effects matrix to speed computation. For more details see the documentation accompanying the package in the lme4 repository at http://www.r-project.org/.
To solve for the maximum likelihood estimates, the design matrix X in equation 2 must be of full column rank. It is well known that this is not the case for a full factorial design matrix with an intercept (as in equation 1), and thus linear combinations (“contrasts”) must be used to define the columns of X in order for the fixed-effect coefficients to be estimable. As our designs are balanced (or nearly balanced), we used orthogonal (or nearly orthogonal) Helmert contrasts between the coefficients associated with light on as compared to light off conditions, terminal stimulation as compared to control conditions, and so on, as reported in the main text. Such contrasts allowed us to compare pooled data (e.g., from several sequential light on vs. light off conditions) against each other within a repeated-measures design—yielding improved parameter estimation and test power while accounting for within-animal correlations.
Results
BLA cells have promiscuous projections throughout the brain, including to the bed nucleus of the stria terminalis (BNST), nucleus accumbens, hippocampus and cortex38, 43. To test whether BLA-CeL synapses could be causally involved in anxiety, it was therefore necessary to develop a method to selectively control BLA terminals in the CeL, without directly affecting other BLA projections. To preferentially target BLA-CeL synapses, we restricted opsin gene expression to BLA glutamatergic projection neurons and restricted light delivery to the CeA. Control of BLA glutamatergic projection neurons was achieved with an adeno-associated virus (AAV5) vector carrying light-activated optogenetic control genes under the control of a CaMKIIα promoter; within the BLA, CaMKIIα is only expressed in glutamatergic pyramidal neurons, not in local interneurons or intercalated cells48. To preferentially deliver light to the CeA projection, virus was delivered unilaterally into the BLA under stereotaxic guidance (
To test the hypothesis that the BLA-CeA pathway could implement an endogenous mechanism for anxiolysis, we probed freely-moving mice under projection-specific optogenetic control in two distinct and well-validated anxiety assays: the elevated plus maze and the open field test (
To determine whether the anxiolytic effect we observed would be specific to activation of BLA terminals in the CeA, and not BLA cells in general, we compared mice receiving projection-specific control (in the ChR2:BLA-CeA group;
We also probed mice on the open field arena for six 3-minute epochs, again testing for reversibility by alternating between no light (off) and light stimulation (on) conditions. Experimental (ChR2:BLA-CeA) mice displayed an immediate, robust, and reversible light-induced anxiolytic response as measured by the time in center of the open field chamber (
We next investigated the physiological basis of this light-induced anxiolytic effect. Glutamatergic neurons in the BLA send robust excitatory projections to CeL neurons as well as to CeM neurons38; however, not only are the CeM synapses distant from the light source (
To confirm the operation of this optogenetically-defined projection, we undertook in vivo experiments, with light delivery protocols matched to those delivered in the behavioral experiments, and activity-dependent immediate early gene (c-fos) expression analysis as the readout to verify the pattern of neuronal activation (
To test the hypothesis that selective illumination of BLA terminals in the CeL induces feed-forward inhibition of CeM output neurons, we combined whole-cell patch-clamp recording with live two-photon imaging to visualize the microcircuit while simultaneously probing the functional relationships among these cells during projection-specific optogenetic control (
To further elucidate the amygdalar microcircuits underlying this anxiolytic effect, we carefully dissected the anatomical and functional properties governing this phenomenon. While some efforts to map the projections of BLA collaterals in the CeA have been made in the rat, we empirically tested whether overlapping or distinct populations of BLA neurons projected to the CeL and CeM (
Next, as our c-fos assays suggested that illumination of BLA terminals in the CeL were sufficient to excite CeL neurons, but not BLA neurons themselves, we sought to confirm this hypothesis with whole-cell recordings. With electrical stimulation, depolarization of axon terminals leads to antidromic spiking at the cell soma. However, there has been evidence that optogenetically-induced depolarization functions via a distinct mechanism. To evaluate the properties of optogenetically-induced terminal stimulation in this amygdalar microcircuit, we recorded from BLA pyramidal neurons expressing ChR2 and moved a light spot (˜120 μm in diameter) in 100 μm steps from the cell soma, both in a direction over a visually-identified axon collateral and in a direction where there was no axon (
Finally, we further explored the mechanism with in vivo pharmacological analysis in the setting of projection-specific optogenetic control. To determine whether the anxiolytic effect we observed could be due to the selective activation of BLA-CeL synapses alone, and not BLA fibers passing through the CeA, nor back-propagation of action potentials to BLA cell bodies which then would innervate all BLA projection target regions, we tested whether local glutamate receptor antagonism would attenuate light-induced anxiolytic effects. This question is of substantial interest since lesions in the CeA that alter anxiety are confounded by the likelihood of ablation of BLA projections to the BNST which pass through CeA6. We unilaterally transduced BLA neurons with AAV-CaMKIIα-ChR2-EYFP and implanted beveled cannulae to implement selective illumination of BLA terminals in the CeA as before (n=8;
In a final series of experiments, to determine if endogenous anxiety-reducing processes could be blocked by selectively inhibiting this pathway, we tested whether the selective inhibition of these optogenetically defined synapses could reversibly increase anxiety. We performed bilateral viral transduction of either eNpHR3.0, a light-activated chloride pump which hyperpolarizes neuronal membranes upon illumination with amber light25, or EYFP alone, both under the CaMKIIα promoter in the BLA, and implanted bilateral beveled guide cannulae to allow selective illumination of BLA terminals in the CeA (
Conclusions: In these experiments, we have identified the BLA-CeL pathway as an endogenous neural substrate for bidirectionally modulating the unconditioned expression of anxiety. While we identify the BLA-CeL pathway as the critical substrate rather than BLA fibers passing through the CeL, it is likely that other downstream circuits, such as CeA projections to the BNST play an important role in the expression of anxiety or anxiety-related behaviors4, 6, 13. Indeed, our findings may support the notion that corticotrophin releasing hormone (CRH) networks in the BNST can be critically involved in modulating anxiety-related behaviors6, 52, as the CeL is a primary source of CRH for the BNST53.
Other neurotransmitters and neuromodulators may modulate or gate effects on distributed neural circuits, including serotonin54, 55, dopamine56, acetylcholine57, glycine58, GABA13 and CRH59. The neural circuitry converging to and diverging from this pathway will provide many opportunities for modulatory control, as parallel or downstream circuits of the BLA-CeL synapse likely contribute to modulate the expression of anxiety phenotypes6, 56. Moreover, upstream of the amygdala, this microcircuit is well-positioned to be recruited by top-down cortical control from regions important for processing fear and anxiety, including the prelimbic, infralimbic and insular cortices that provide robust innervation to the BLA and CeL.4, 13, 23, 60.
Our examination of the BLA anatomy suggests that the populations of BLA neurons projecting to CeL and CeM neurons are largely non-overlapping. In natural states, the CeL-projecting BLA neurons may excite CeM-projecting BLA neurons in a microcircuit homeostatic mechanism This may also represent a potential mechanism underlying anxiety disorders, when there are synaptic changes that skew the balance of the circuit to allow uninhibited CeM activation.
Together, the data presented here support identification of the BLA-CeL synapse as a critical circuit element both necessary and sufficient for the expression of endogenous anxiolysis in the mammalian brain, providing a novel source of insight into anxiety as well as a new kind of treatment target, and demonstrate the importance of resolving specific projections in the study of neural circuit function relevant to psychiatric disease.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention.
All references, publications, and patent applications disclosed herein are hereby incorporated by reference in their entirety.
This application claims the priority benefit of U.S. provisional application Ser. Nos. 61/410,748 filed on Nov. 5, 2010, and 61/464,806 filed on Mar. 8, 2011, the contents of each of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2011/059298 | 11/4/2011 | WO | 00 | 7/29/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/061690 | 5/10/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 2968302 | Fry et al. | Jan 1961 | A |
| 3131690 | Innis et al. | May 1964 | A |
| 3499437 | Balamuth et al. | Mar 1970 | A |
| 3567847 | Price | Mar 1971 | A |
| 4343301 | Indech | Aug 1982 | A |
| 4559951 | Dahl et al. | Dec 1985 | A |
| 4616231 | Autrey et al. | Oct 1986 | A |
| 4865042 | Umemura et al. | Sep 1989 | A |
| 4879284 | Land et al. | Nov 1989 | A |
| 5032123 | Katz et al. | Jul 1991 | A |
| 5041224 | Ohyama et al. | Aug 1991 | A |
| 5082670 | Gage et al. | Jan 1992 | A |
| 5249575 | Di Mino et al. | Oct 1993 | A |
| 5267152 | Yang et al. | Nov 1993 | A |
| 5290280 | Daikuzono et al. | Mar 1994 | A |
| 5330515 | Rutecki et al. | Jul 1994 | A |
| 5460950 | Barr et al. | Oct 1995 | A |
| 5460954 | Lee et al. | Oct 1995 | A |
| 5470307 | Lindall | Nov 1995 | A |
| 5495541 | Murray et al. | Feb 1996 | A |
| 5520188 | Hennige et al. | May 1996 | A |
| 5527695 | Hodges et al. | Jun 1996 | A |
| 5550316 | Mintz | Aug 1996 | A |
| 5641650 | Turner et al. | Jun 1997 | A |
| 5703985 | Owyang et al. | Dec 1997 | A |
| 5722426 | Kolff | Mar 1998 | A |
| 5738625 | Gluck | Apr 1998 | A |
| 5739273 | Engelman et al. | Apr 1998 | A |
| 5741316 | Chen et al. | Apr 1998 | A |
| 5755750 | Petruska et al. | May 1998 | A |
| 5756351 | Isacoff et al. | May 1998 | A |
| 5782896 | Chen et al. | Jul 1998 | A |
| 5795581 | Segalman et al. | Aug 1998 | A |
| 5807285 | Vaitekunas et al. | Sep 1998 | A |
| 5939320 | Littman et al. | Aug 1999 | A |
| 6134474 | Fischell et al. | Oct 2000 | A |
| 6161045 | Fischell et al. | Dec 2000 | A |
| 6253109 | Gielen | Jun 2001 | B1 |
| 6303362 | Kay et al. | Oct 2001 | B1 |
| 6334846 | Ishibashi et al. | Jan 2002 | B1 |
| 6336904 | Nikolchev | Jan 2002 | B1 |
| 6364831 | Crowley | Apr 2002 | B1 |
| 6377842 | Pogue et al. | Apr 2002 | B1 |
| 6436708 | Leone et al. | Aug 2002 | B1 |
| 6473639 | Fischell et al. | Oct 2002 | B1 |
| 6480743 | Kirkpatrick et al. | Nov 2002 | B1 |
| 6489115 | Lahue et al. | Dec 2002 | B2 |
| 6497872 | Weiss et al. | Dec 2002 | B1 |
| 6506154 | Ezion et al. | Jan 2003 | B1 |
| 6536440 | Dawson | Mar 2003 | B1 |
| 6551346 | Crossley | Apr 2003 | B2 |
| 6567690 | Giller et al. | May 2003 | B2 |
| 6597954 | Pless et al. | Jul 2003 | B1 |
| 6609020 | Gill | Aug 2003 | B2 |
| 6615080 | Unsworth et al. | Sep 2003 | B1 |
| 6631283 | Storrie et al. | Oct 2003 | B2 |
| 6632672 | Calos | Oct 2003 | B2 |
| 6647296 | Fischell et al. | Nov 2003 | B2 |
| 6685656 | Duarte et al. | Feb 2004 | B1 |
| 6686193 | Maher et al. | Feb 2004 | B2 |
| 6721603 | Zabara et al. | Apr 2004 | B2 |
| 6729337 | Dawson | May 2004 | B2 |
| 6780490 | Tanaka et al. | Aug 2004 | B1 |
| 6790652 | Terry et al. | Sep 2004 | B1 |
| 6790657 | Arya | Sep 2004 | B1 |
| 6805129 | Pless et al. | Oct 2004 | B1 |
| 6810285 | Pless et al. | Oct 2004 | B2 |
| 6889085 | Dawson | May 2005 | B2 |
| 6921413 | Mahadevan-Jansen et al. | Jul 2005 | B2 |
| 6969449 | Maher et al. | Nov 2005 | B2 |
| 6974448 | Petersen | Dec 2005 | B2 |
| 7045344 | Kay et al. | May 2006 | B2 |
| 7091500 | Schnitzer | Aug 2006 | B2 |
| 7144733 | Miesenbock et al. | Dec 2006 | B2 |
| 7175596 | Vitek et al. | Feb 2007 | B2 |
| 7191018 | Gielen et al. | Mar 2007 | B2 |
| 7211054 | Francis et al. | May 2007 | B1 |
| 7220240 | Struys et al. | May 2007 | B2 |
| 7298143 | Jaermann et al. | Nov 2007 | B2 |
| 7313442 | Velasco et al. | Dec 2007 | B2 |
| 7603174 | De Ridder | Oct 2009 | B2 |
| 7610100 | Jaax et al. | Oct 2009 | B2 |
| 7613520 | De Ridder | Nov 2009 | B2 |
| 7686839 | Parker | Mar 2010 | B2 |
| 7824869 | Hegemann et al. | Nov 2010 | B2 |
| 7988688 | Webb et al. | Aug 2011 | B2 |
| 8386312 | Pradeep et al. | Feb 2013 | B2 |
| 8398692 | Deisseroth et al. | Mar 2013 | B2 |
| 8401609 | Deisseroth et al. | Mar 2013 | B2 |
| 20020094516 | Calos et al. | Jul 2002 | A1 |
| 20020155173 | Chopp et al. | Oct 2002 | A1 |
| 20020164577 | Tsien et al. | Nov 2002 | A1 |
| 20030009103 | Yuste et al. | Jan 2003 | A1 |
| 20030026784 | Koch et al. | Feb 2003 | A1 |
| 20030040080 | Miesenbock et al. | Feb 2003 | A1 |
| 20030050258 | Calos | Mar 2003 | A1 |
| 20030097122 | Ganz et al. | May 2003 | A1 |
| 20030104512 | Freeman et al. | Jun 2003 | A1 |
| 20030125719 | Furnish | Jul 2003 | A1 |
| 20030204135 | Bystritsky | Oct 2003 | A1 |
| 20030232339 | Shu et al. | Dec 2003 | A1 |
| 20040034882 | Vale et al. | Feb 2004 | A1 |
| 20040039312 | Hillstead et al. | Feb 2004 | A1 |
| 20040122475 | Myrick et al. | Jun 2004 | A1 |
| 20040203152 | Calos | Oct 2004 | A1 |
| 20050058987 | Shi et al. | Mar 2005 | A1 |
| 20050119315 | Fedida et al. | Jun 2005 | A1 |
| 20050124897 | Chopra | Jun 2005 | A1 |
| 20050153885 | Yun et al. | Jul 2005 | A1 |
| 20050197679 | Dawson | Sep 2005 | A1 |
| 20050202398 | Hegemann et al. | Sep 2005 | A1 |
| 20050215764 | Tuszynski et al. | Sep 2005 | A1 |
| 20050240127 | Seip et al. | Oct 2005 | A1 |
| 20050267011 | Deisseroth et al. | Dec 2005 | A1 |
| 20050267454 | Hissong et al. | Dec 2005 | A1 |
| 20060025756 | Francischelli et al. | Feb 2006 | A1 |
| 20060034943 | Tuszynski | Feb 2006 | A1 |
| 20060057192 | Kane | Mar 2006 | A1 |
| 20060058671 | Vitek et al. | Mar 2006 | A1 |
| 20060058678 | Vitek et al. | Mar 2006 | A1 |
| 20060100679 | DiMauro et al. | May 2006 | A1 |
| 20060106543 | Deco et al. | May 2006 | A1 |
| 20060155348 | de Charms | Jul 2006 | A1 |
| 20060161227 | Walsh et al. | Jul 2006 | A1 |
| 20060184069 | Vaitekunas | Aug 2006 | A1 |
| 20060190044 | Libbus et al. | Aug 2006 | A1 |
| 20060206172 | DiMauro et al. | Sep 2006 | A1 |
| 20060216689 | Maher et al. | Sep 2006 | A1 |
| 20060236525 | Sliwa et al. | Oct 2006 | A1 |
| 20060241697 | Libbus et al. | Oct 2006 | A1 |
| 20060253177 | Taboada et al. | Nov 2006 | A1 |
| 20060271024 | Gertner et al. | Nov 2006 | A1 |
| 20070031924 | Li et al. | Feb 2007 | A1 |
| 20070054319 | Boyden et al. | Mar 2007 | A1 |
| 20070060915 | Kucklick | Mar 2007 | A1 |
| 20070135875 | Demarais et al. | Jun 2007 | A1 |
| 20070156180 | Jaax et al. | Jul 2007 | A1 |
| 20070191906 | Lyer et al. | Aug 2007 | A1 |
| 20070196838 | Chesnut et al. | Aug 2007 | A1 |
| 20070197918 | Vitek et al. | Aug 2007 | A1 |
| 20070219600 | Gertner et al. | Sep 2007 | A1 |
| 20070220628 | Glassman et al. | Sep 2007 | A1 |
| 20070239080 | Schaden et al. | Oct 2007 | A1 |
| 20070239210 | Libbus et al. | Oct 2007 | A1 |
| 20070253995 | Hildebrand et al. | Nov 2007 | A1 |
| 20070261127 | Boyden et al. | Nov 2007 | A1 |
| 20070282404 | Cottrell et al. | Dec 2007 | A1 |
| 20070295978 | Coushaine et al. | Dec 2007 | A1 |
| 20080020465 | Padidam | Jan 2008 | A1 |
| 20080027505 | Levin et al. | Jan 2008 | A1 |
| 20080033569 | Ferren et al. | Feb 2008 | A1 |
| 20080046053 | Wagner et al. | Feb 2008 | A1 |
| 20080050770 | Zhang et al. | Feb 2008 | A1 |
| 20080051673 | Kong et al. | Feb 2008 | A1 |
| 20080060088 | Shin et al. | Mar 2008 | A1 |
| 20080065158 | Ben-Ezra et al. | Mar 2008 | A1 |
| 20080065183 | Whitehurst et al. | Mar 2008 | A1 |
| 20080077200 | Bendett et al. | Mar 2008 | A1 |
| 20080085265 | Schneider et al. | Apr 2008 | A1 |
| 20080103551 | Masoud | May 2008 | A1 |
| 20080119421 | Tuszynski et al. | May 2008 | A1 |
| 20080125836 | Streeter et al. | May 2008 | A1 |
| 20080167261 | Sclimenti | Jul 2008 | A1 |
| 20080175819 | Kingsman et al. | Jul 2008 | A1 |
| 20080176076 | Van Veggel et al. | Jul 2008 | A1 |
| 20080200749 | Zheng et al. | Aug 2008 | A1 |
| 20080221452 | Njemanze | Sep 2008 | A1 |
| 20080227139 | Deisseroth et al. | Sep 2008 | A1 |
| 20080228244 | Pakhomov et al. | Sep 2008 | A1 |
| 20080262411 | Dobak | Oct 2008 | A1 |
| 20080287821 | Jung et al. | Nov 2008 | A1 |
| 20080290318 | Van Veggel et al. | Nov 2008 | A1 |
| 20090030930 | Pradeep et al. | Jan 2009 | A1 |
| 20090069261 | Dodge et al. | Mar 2009 | A1 |
| 20090088680 | Deisseroth et al. | Apr 2009 | A1 |
| 20090093403 | Zhang et al. | Apr 2009 | A1 |
| 20090099038 | Deisseroth et al. | Apr 2009 | A1 |
| 20090112133 | Deisseroth et al. | Apr 2009 | A1 |
| 20090118800 | Deisseroth et al. | May 2009 | A1 |
| 20090148861 | Pegan et al. | Jun 2009 | A1 |
| 20090157145 | Cauller | Jun 2009 | A1 |
| 20090254134 | Nikolov et al. | Oct 2009 | A1 |
| 20090268511 | Birge et al. | Oct 2009 | A1 |
| 20090319008 | Mayer | Dec 2009 | A1 |
| 20090326603 | Boggs | Dec 2009 | A1 |
| 20100009444 | Herlitze et al. | Jan 2010 | A1 |
| 20100016783 | Bourke et al. | Jan 2010 | A1 |
| 20100145418 | Zhang et al. | Jun 2010 | A1 |
| 20100190229 | Deisseroth et al. | Jul 2010 | A1 |
| 20100234273 | Deisseroth et al. | Sep 2010 | A1 |
| 20110021970 | Vo-Dinh et al. | Jan 2011 | A1 |
| 20110092800 | Yoo et al. | Apr 2011 | A1 |
| 20110105998 | Deisseroth et al. | May 2011 | A1 |
| 20110112179 | Deisseroth et al. | May 2011 | A1 |
| 20110125077 | Denison et al. | May 2011 | A1 |
| 20110125078 | Denison et al. | May 2011 | A1 |
| 20110159562 | Deisseroth et al. | Jun 2011 | A1 |
| 20110166632 | Deisseroth et al. | Jul 2011 | A1 |
| 20110172653 | Deisseroth et al. | Jul 2011 | A1 |
| 20110301529 | Deisseroth et al. | Dec 2011 | A1 |
| 20110311489 | Deisseroth et al. | Dec 2011 | A1 |
| 20120093772 | Horsager et al. | Apr 2012 | A1 |
| 20120165904 | Deisseroth et al. | Jun 2012 | A1 |
| 20120253261 | Poletto et al. | Oct 2012 | A1 |
| 20130019325 | Deisseroth et al. | Jan 2013 | A1 |
| 20130144359 | Kishawi et al. | Jun 2013 | A1 |
| Number | Date | Country |
|---|---|---|
| 1 334 748 | Aug 2003 | EP |
| 2006- 295350 | Oct 1994 | JP |
| WO 0027293 | May 2000 | WO |
| WO 01-25466 | Apr 2001 | WO |
| WO 03106486 | Feb 2003 | WO |
| WO 03-040323 | May 2003 | WO |
| WO 03-084994 | Oct 2003 | WO |
| WO 03-102156 | Dec 2003 | WO |
| WO 2007-024391 | Mar 2007 | WO |
| WO 2007-131180 | Nov 2007 | WO |
| WO 2008106694 | Sep 2008 | WO |
| WO2009119782 | Oct 2009 | WO |
| WO 2009-131837 | Oct 2009 | WO |
| WO 2010011404 | Jan 2010 | WO |
| WO 2010056970 | May 2010 | WO |
| WO-2010123993 | Oct 2010 | WO |
| WO 2011066320 | Jun 2011 | WO |
| WO 2011-116238 | Sep 2011 | WO |
| WO 2011127088 | Oct 2011 | WO |
| WO 2012061676 | May 2012 | WO |
| WO2012061681 | May 2012 | WO |
| WO2012061684 | May 2012 | WO |
| WO2012061688 | May 2012 | WO |
| WO2012061690 | May 2012 | WO |
| WO 2012061744 | May 2012 | WO |
| WO 2012134704 | Oct 2012 | WO |
| WO 2013126521 | Aug 2013 | WO |
| WO 2013142196 | Sep 2013 | WO |
| Entry |
|---|
| Hikida et al., “Increased sensitivity to cocaine by cholinergic cell ablation in nucleus accumbens”, PNAS, Nov. 2001, 98(23): 13351-13354. |
| Hikida et al., “Acetylcholine enhancement in the nucleus accumbens prevents addictive behaviors of cocaine and morphine”, PNAS, May 2003, 100(10):6169-6173. |
| Kitabatake et al., “Impairment of reward-related learning by cholinergic cell ablation in the striatum”, PNAS, Jun. 2003, 100(13):7965-7970. |
| Tamai, “Progress in Pathogenesis and Therapeutic Research in Retinitis Pigmentosa and Age Related Macular Degeneration”, Nippon Ganka Gakkai Zasshi, vol. 108, No. 12, Dec. 2004, pp. 750-769. |
| Cazillis et al., “VIP and PACAP induce selective neuronal differentiation of mouse embryonic stem cells”, Eur J Neurosci, 2004, 19(4):798-808. |
| Morelli et al., “Neuronal and glial cell type-specific promoters within adenovirus recombinants restrict the expression of the apoptosis-inducing molecule Fas ligand to predetermined brain cell types, and abolish peripheral liver toxicity”, Journal of General Virology, 1999, 80:571-583. |
| Berke, et al. “Addiction, Dopamine, and the Molecular Mechanisms of Memory”, Molecular Plasticity, 2000, vol. 25: pp. 515-532. |
| Goshen et al. “Dynamics of Retrieval Strategies for Remote Memories”, Cell, 2011, vol. 147: pp. 678-589. |
| Jimenez S.A & Maren S. et al/ “Nuclear disconnection within the amygdala reveals a direct pathway to fear”, Learning Memory, 2009, vol. 16: pp. 766-768. |
| Ehrlich I. et al. “Amygdala inhibitory circuits and the control of fear memory”, Neuron, 2009. Friedrich Meischer Institute, vol. 62: pp. 757-771. |
| Berndt et al. “Bi-stable neural state switches”, Nature Neuroscience, 2009, vol. 12, No. 2: pp. 229-234. |
| Simmons et al. “Localization and function of NK3 subtype Tachykinin receptors of layer pyramidal neurons of the guinea-pig medial prefrontal cortex”, Neuroscience, 2008, vol. 156, No. 4: pp. 987-994. |
| Aebischer, et al. “Long-Term Cross-Species Brain Transplantation of a Polymer-Encapsulated Dopamine-Secreting Cell Line”, Experimental Neurology, 1991, vol. 111, pp. 269-275. |
| Ahmad, et al. “The Drosophila rhodopsin cytoplasmic tail domain is required for maintenance of rhabdomere structure.” The FASEB Journal, 2007, vol. 21, p. 449-455. |
| Airan, et al., “Temporally Precise in vivo Control of Intracellular Signaling”, 2009, Nature, vol. 458, No. 7241, pp. 1025-1029. |
| Akirav, et al. “The role of the medial prefrontal cortex-amygdala circuit in stress effects on the extinction of fear”, Neural Plasticity, 2007: vol. 2007 Article ID:30873, pp. 1-11. |
| Ang, et at. “Hippocampal CA1 Circuitry Dynamically Gates Direct Cortical Inputs Preferentially at Theta Frequencies.” The Journal of Neurosurgery, 2005, vol. 25, No. 42, pp. 9567-9580. |
| Araki, et al. “Site-Directed Integration of the cre Gene Mediated by Cre Recombinase Using a Combination of Mutant lox Sites”, Nucleic Acids Research, 2002, vol. 30, No. 19, pp. 1-8. |
| Aravanis, et al. “An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology,” J. Neural. Eng., 2007, vol. 4(3):S143-S156. |
| Argos, et al. “The integrase family of site-specific recombinases: regional similarities and global diversity”, The EMBO Journal, 1986, vol. 5, No. 2, pp. 433-440. |
| Bamberg et al. “Light-driven proton or chloride pumping by halorhodopsin.” Proc. Natl. Academy Science USA, 1993, vol. 90, No. 2, p. 639-643. |
| Banghart, et al. “Light-activated ion channels for remote control of neuronal firing”. Nature Neuroscience, 2004, vol. 7, No. 12 pp. 1381-1386. |
| Basil et al. “Is There Evidence for Effectiveness of Transcranial Magnetic Stimulation in the Treatment of Psychiatric Disorders?” Psychiatry, 2005, pp. 64-69. |
| Bebbington et al., “The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning” vol. 3, Academic Press, New York, 1987. |
| Benabid “Future strategies to restore brain functions,” Conference proceedings from Medicine Meets Millennium: World Congress of Medicine and Health, 2000, 6 pages. |
| Benoist et al. “In vivo sequence requirements of the SV40 early promotor region” Nature (London), 1981, vol. 290(5804): pp. 304-310. |
| Berges et al., “Transduction of Brain by Herpes Simplex Virus Vectors”, Molecular Therapy, 2007, vol. 15, No. 1: pp. 20-29. |
| Berridge et al., “The Versatility and Universality of Calcium Signaling”, Nature Reviews: Molecular Cell Biology, 2000, vol. 1: pp. 11-21. |
| Bocquet et al. “A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family.” Nature Letters, 2007, vol. 445, p. 116-119. |
| Boyden, et al. “Millisecond-timescale, genetically targeted optical control of neural activity” Nature Neuroscience, 2005, vol. 8, No. 9: pp. 1263-1268. |
| Bi, et al. “Ectopic Expression of a Microbial-Type Rhodopsin Restores Visual Responses in Mice with Photoreceptor Degeneration”, Neuron, 2006, vol. 50, No. 1: pp. 23-33. |
| Bi, et al. “Synaptic Modifications in Cultured Hippocampal Neurons: Dependence on Spike Timing, Synaptic Strength, and Postsynaptic Cell Type”, Journal of Neuroscience, 1998, vol. 18, No. 24: pp. 10464-10472. |
| Blomer et al., “Highly Efficient and Sustained Gene Transfer in Adult Neurons with Lentivirus Vector”, Journal of Virology,1997, vol. 71, No. 9: pp. 6641-6649. |
| Braun, “Two Light-activated Conductances in the Eye of the Green Alga Volvox carteri”, 1999, Biophys J., vol. 76, No. 3, pp. 1668-1678. |
| Brinton, et al. “Preclinical analyses of the therapeutic potential of allopregnanolone to promote neurogenesis in vitro and in vivo in transgenic mouse model of Alzheimer's disease.” Current Alzheimer Research, 2006, vol. 3, No. 1: pp. 11-17. |
| Brosenitsch et al, “Physiological Patterns of Electrical Stimulation Can Induce Neuronal Gene Expression by Activating N-Type Calcium Channels,” Journal of Neuroscience, 2001, vol. 21, No. 8, pp. 2571-2579. |
| Brown, et al. “Long-term potentiation induced by θ frequency stimulation is regulated by a protein phosphate-operated gate.” The Journal of Neuroscience, 2000, vol. 20, No. 21, pp. 7880-7887. |
| Callaway, et al. “Photostimulation using caged glutamate reveals functional circuitry in living brain slices”, Proc. Natl. Acad. Sci. USA., 1993, vol. 90: pp. 7661-7665. |
| Campagnola et al. “Fiber-coupled light-emitting diode for localized photostimulation of neurons expressing channelrhodopsin-2.” Journal of Neuroscience Methods , 2008, vol. 169, Issue 1. Abstract only. |
| Cardin, et al. “Driving Fast spiking Cells Induces Gamma Rhythm and Controls Sensory Responses”, 2009, Nature, vol. 459, vol. 7247, pp. 663-667. |
| Cenatiempo “Prokaryotic gene expression in vitro: transcription-translation coupled systems”, Biochimie, 1986, vol. 68(4): pp. 505-515. |
| Claudio et al. “Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit.” PNAS USA,1983, vol. 80, p. 1111-1115. |
| Collingridge et al. “Inhibitory post-synaptic currents in rat hippocampal CA1 neurones.” J. Physiol., 1984, vol. 356, pp. 551-564. |
| Covington, et al. “Antidepressant Effect of Optogenetic Stimulation of the Medial Prefrontal Cortex.” Journal of Neuroscience, 2010, vol. 30(48), pp. 16082-16090. |
| Crouse, et al. “Expression and amplification of engineered mouse dihydrofolate reductase minigenes” Mol. Cell. Biol. , 1983, vol. 3(2): pp. 257-266. |
| Cucchiaro et al., “Phaseolus vulgaris leucoagglutinin (PHA-L): a neuroanatomical tracer for electron microscopic analysis of synaptic circuitry in the cat's dorsal lateral geniculate nucleus” J. Electron. Microsc. Tech., 1990, 15 (4):352-368. |
| Cucchiaro et al., “Electron-Microsoft Analysis of Synaptic Input from the Perigeniculate Nucleus to A-Lamine of the Lateral Geniculate Nucleus in Cats”, The Journal of Comparitive Neurology, 1991, vol. 310, pp. 316-336. |
| Cui, et al., “Electrochemical deposition and characterization of conducting polymer polypyrrole/PSS on multichannel neural probes,” Sensors and Actuators, 2001, vol. 93(1): pp. 8-18. |
| Date, et al. “Grafting of Encapsulated Dopamine-Secreting Cells in Parkinson's Disease: Long-Term Primate Study”, Cell Transplant, 2000, vol. 9, pp. 705-709. |
| Dalva, et al. “Rearrangements of Synaptic Connections in Visual Cortex Revealed by Laser Photostimulation”, Science, 1994,vol. 265, pp. 255-258. |
| Dederen, et al. “Retrograde neuronal tracing with cholera toxin B subunit: comparison of three different visualization methods”, Histochemical Journal, 1994, vol. 26, pp. 856-862. |
| De Foubert et al. “Fluoxetine-Induced Change in Rat Brain Expression of Brain-Derived Neurotrophic Factor Varies Depending on Length of Treatment,” Neuroscience, 2004, vol. 128, pp. 597-604. |
| Deisseroth et al., “Signaling from Synapse to Nucleus: Postsynaptic CREB Phosphorylation During Multiple Forms of Hippocampal Synaptic Plasticity”, Neuron, 1996, vol. 16, pp. 89-101. |
| Deisseroth et al., “Translocation of Calmodulin to the Nucleus Supports CREB Phosphorylation in Hippocampal Neurons”, Nature, 1998, vol. 392, pp. 198-202. |
| Deisseroth et al., “Signaling from Synapse to Nucleus: the logic Behind the Mechanisms”, Currrent Opinion in Neurobiology, 2003, vol. 13, pp. 354-365. |
| Deisseroth et al., “Excitation-neurogenesis Coupling in Adult Neural Stem/Progenitor Cells”, 2004, Neuron, vol. 42, pp. 535-552. |
| Deisseroth “Next-generation optical technologies for illuminating genetically targeted brain circuits,” The Journal of Neuroscience, 2006, vol. 26, No. 41, pp. 10380-10386. |
| Denk, W., et al. “Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy”, Journal of Neuroscience Methods, 1994, vol. 54, pp. 151-162. |
| Ditterich, et al. “Microstimulation of visual cortex affects the speed of perceptual decisions”, 2003, Nature Neuroscience, vol. 6, No. 8, pp. 891-898. |
| Dittgen, et al. “Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo”, PNAS, 2004, vol. 10I, No. 52, pp. 18206-18211. |
| Emerich, et al. “A Novel Approach to Neural Transplantation in Parkinson's Disease: Use of Polymer-Encapsulated Cell Therapy”, Neuroscience and Biobehavioral Reviews, 1992, vol. 16, pp. 437-447. |
| Ensell, et al. “Silicon-based microelectrodes for neurophysiology, micromachined from silicon-on-insulator wafers,” Med. Biol. Eng. Comput., 2000, vol. 38, pp. 175-179. |
| Eisen, “Treatment of amyotrophic lateral sclerosis”, Drugs Aging, 1999; vol. 14, No. 3, pp. 173-196. |
| Ernst, et al. “Photoactivation of Channelrhodopsin”, 2008, vol. 283, No. 3, pp. 1637-1643. |
| Evanko “Optical excitation yin and yang” Nature Methods, 2007, 4:384. |
| Esposito et al. “The integrase family of tyrosine recombinases: evolution of a conserved active site domain”, Nucleic Acids Research, 1997, vol. 25, No. 18, pp. 3605-3614. |
| Fabian et al. “Transneuronal transport of lectins” Brain Research, 1985, vol. 344, pp. 41-48. |
| Falconer et al. “High-throughput screening for ion channel modulators,” Journal of Biomolecular Screening, 2002, vol. 7, No. 5, pp. 460-465. |
| Farber, et al. “Identification of Presynaptic Neurons by Laser Photostimulation”, Science, 1983, vol. 222, pp. 1025-1027. |
| Feng, et al. “Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP”, Neuron, 2000, vol. 28, pp. 41-51. |
| Fisher, J. et al. “Spatiotemporal Activity Patterns During Respiratory Rhythmogenesis in the Rat Ventrolateral Medulla,” The Journal of Neurophysiol, 2006, vol. 95, pp. 1982-1991. |
| Fitzsimons et al., “Promotors and Regulatory Elements that Improve Adeno-Associated Virus Transgene Expression in the Brain”, 2002, Methods, vol. 28, pp. 227-236. |
| Foster, “Bright blue times”, Nature, 2005, vol. 433, pp. 698-699. |
| Genbank Accession No. DQ094781 (Jan. 15, 2008). |
| Gelvich et al. “Contact flexible microstrip applicators (CFMA) in a range from microwaves up to short waves,” IEEE Transactions on Biomedical Engineering, 2002, vol. 49, Issue 9: 1015-1023. |
| Gigg, et al. “Glutamatergic hippocampal formation projections to prefrontal cortex in the rat are regulated by GABAergic inhibition and show convergence with glutamatergic projections from the limbic thalamus,” Hippocampus, 1994, vol. 4, No. 2, pp. 189-198. |
| Gilman, et al. “Isolation of sigma-28-specific promoters from Bacillus subtilis DNA” Gene, 1984, vol. 32(1-2): pp. 11-20. |
| Glick et al. “Factors affecting the expression of foreign proteins in Escherichia coli”, Journal of Industrial Microbiology, 1987, vol. 1(5): pp. 277-282. |
| Goekoop, R. et al. “Cholinergic challenge in Alzheimer patients and mild cognitive impairment differentially affects hippocampal activation-a pharmacological fMRI study.” Brain, 2006, vol. 129, pp. 141-157. |
| Gold, et al. “Representation of a perceptual decision in developing oculomotor commands”, Nature, 2000, vol. 404, pp. 390-394. |
| Gonzalez, et al., “Cell-Based Assays and Instrumentation for Screening Ion-Channel Targets”, DDT, 1999, vol. 4, No. 9, pp. 431-439. |
| Gordon, et al. “Regulation of Thy—1 Gene Expression in Transgenic Mice”, Cell, 1987, vol. 50, pp. 445-452. |
| Gorelova et al., “The course of neural projection from the prefrontal cortex to the nucleus accumbens in the rat”, Neuroscience, 1997, vol. 76, No. 3, pp. 689-706. |
| Gottesman et al.“Bacterial regulation: global regulatory networks,” Ann. Rev. Genet. , 1984, vol. 18, pp. 415-441. |
| Gradinaru, et al. “ENpHR: a Natronomonas Halorhodopsin Enhanced for Optogenetic Applications”, 2008, Brain Cell Biol., vol. 36 (1-4), pp. 129-139. |
| Greenberg, et al. “Three-year outcomes in deep brain stimulation for highly resistant obsessive-compulsive disorder,” Neuropsychopharmacology, 2006, vol. 31, pp. 2384-2393. |
| Gregory, et al. “Integration site for Streptomyces phage φBT1 and development of site-specific integrating vectors”, Journal of Bacteriology, 2003, vol. 185, No. 17, pp. 5320-5323. |
| Groth et al. “Phage integrases: biology and applications,” Journal of Molecular Biology, 2004, vol. 335, pp. 667-678. |
| Groth, et al. “A phage integrase directs efficient site-specific integration in human cells”, PNAS, 2000, vol. 97, No. 11, pp. 5995-6000. |
| Guatteo, et al. “Temperature sensitivity of dopaminergic neurons of the substantia nigra pars compacta: Involvement of transient receptor potential channels,” Journal of Neurophysiol. , 2005, vol. 94, pp. 3069-3080. |
| Gulick, et al. “Transfection using DEAE-Dextran” Supplement 40, Current Protocols in Molecular Biology, 1997, Supplement 40, 9.2.1-9.2.10. |
| Gur et al., “A Dissociation Between Brain Activity and Perception: Chromatically Opponent Cortical Neurons Signal Chromatic Flicker that is not Perceived”, Vision Research, 1997, vol. 37, No. 4, pp. 377-382. |
| Hallet et al. “Transposition and site-specific recombination: adapting DNA cut-and-paste mechanisms to a variety of genetic rearrangements,” FEMS Microbiology Reviews, 1997, vol. 21, No. 2, pp. 157-178. |
| Hamer, et al. “Regulation In Vivo of a cloned mammalian gene: cadmium induces the transcription of a mouse metallothionein gene in SV40 vectors,” Journal of Molecular Applied Genetics, 1982, vol. 1, No. 4, pp. 273-288. |
| Hausser, et al. “Tonic Synaptic Inhibition Modulates Neuronal Output Pattern and Spatiotemporal Synaptic Integration”, Neuron, 1997, vol. 19, pp. 665-678. |
| Hegemann et al., “All-trans Retinal Constitutes the Functional Chromophore in Chlamydomonas rhodopsin”, Biophys. J. , 1991, vol. 60, pp. 1477-1489. |
| Herlitze, et al., “New Optical Tools for Controlling Neuronal Activity”, 2007, Curr Opin Neurobiol, vol. 17, No. 1, pp. 87-94. |
| Herry, et al. “Switching on and off fear by distinct neuronal circuits,” Nature, 2008, vol. 454, pp. 600-606. |
| Hildebrandt et al, “Bacteriorhodopsin expressed in Schizosaccharomyces pombe pumps protons through the plasma membrane,” PNAS, 1993, vol. 90, pp. 3578-3582. |
| Hirase, et al. “Multiphoton stimulation of neurons”, J Neurobiol, 2002, vol. 5I, No. 3: pp. 237-247. |
| Hodaie, et al., “Chronic Anterior Thalamus Stimulation for Intractable Epilepsy,” Epilepsia, 2002, vol. 43, pp. 603-608. |
| Hoffman et al., “K+ Channel Regulation of Signal Propagation in Dendrites of Hippocampal Pyramidal Neurons”, 1997, Nature, vol. 387: pp. 869-874. |
| Hosokawa, T. et al. “Imaging spatio-temporal patterns of long-term potentiation in mouse hippocampus.” Philos. Trans. R. Soc. Lond. B., 2003, vol. 358, pp. 689-693. |
| Hynynen, et al. “Clinical applications of focused ultrasound—The brain.” Int. J. Hyperthermia, 2007, vol. 23, No. 2: pp. 193-202. |
| International Search Report for International Application No. PCT/US2009/053474, dated Oct. 8, 2009. |
| Isenberg et al. “Cloning of a Putative Neuronal Nicotinic Aceylcholine Receptor Subunit,” Journal of Neurochemistry, 1989, pp. 988-991. |
| Jekely, “Evolution of Phototaxis”, 2009, Phil. Trans. R. Soc. B, vol. 364, pp. 2795-2808. |
| Johansen, et al., “Optical Activation of Lateral Amygdala Pyramidal Cells Instructs Associative Fear Learning”, 2010, PNAS, vol. 107, No. 28, pp. 12692-12697. |
| Johnston et al. “Isolation of the yeast regulatory gene GAL4 and analysis of its dosage effects on the galactose/melibiose regulon,” PNAS, 1982, vol. 79, pp. 6971-6975. |
| Kandel, E.R., et al. “Electrophysiology of Hippocampal Neurons: I. Sequential Invasion and Synaptic Organization,” J Neurophysiol, 1961, vol. 24, pp. 225-242. |
| Kandel, E.R., et al. “Electrophysiology of Hippocampal Neurons: II. After-Potentials and Repetitive Firing”, J Neurophysiol., 1961, vol. 24, pp. 243-259. |
| Karreman et al. “On the use of double FLP recognition targets (FRTs) in the LTR of retroviruses for the construction of high producer cell lines”, Nucleic Acids Research, 1996, vol. 24, No. 9: pp. 1616-1624. |
| Kato et al. “Present and future status of noninvasive selective deep heating using RF in hyperthermia.” Med & Biol. Eng. & Comput 31 Supp: S2-11, 1993. Abstract. p. S2 only. |
| Katz, et al. “Scanning laser photostimulation: a new approach for analyzing brain circuits,” Journal of Neuroscience Methods, 1994, vol. 54, pp. 205-218. |
| Khodakaramian, et al. “Expression of Cre Recombinase during Transient Phage Infection Permits Efficient Marker Removal in Streptomyces,” Nucleic Acids Research, 2006, vol. 34, No. 3:e20, pp. 1-5. |
| Khossravani et al., “Voltage-Gated Calcium Channels and Idiopathic Generalized Epilepsies”, Physiol. Rev., 2006, vol. 86: pp. 941-966. |
| Kianianmomeni, et al. “Channelrhodopsins of Volvox carteri are Photochromic Proteins that are Specifically Expressed in Somatic Cells under Control of Light, Temperature, and the Sex Inducer”, 2009, Plant Physiology, vol. 151, No. 1, pp. 347-366. |
| Kim et al., “Light-Driven Activation of β2-Adrenergic Receptor Signaling by a Chimeric Rhodopsin Containing the β2-Adrenergic Receptor Cytoplasmic Loops,” Biochemistry, 2005, vol. 44, No. 7, pp. 2284-2292. |
| Kingston et al. “Transfection of DNA into Eukaryotic Cells,” Supplement 63, Current Protocols in Molecular Biology, 1996, 9.1.1-9.1.11, 11 pages. |
| Kingston et al. “Transfection and Expression of Cloned DNA,” Supplement 31, Current Protocols in Immunology, 1999, 10.13.1-10.13.9. |
| Kita, H. et al. “Effects of dopamine agonists and antagonists on optical responses evoked in rat frontal cortex slices after stimulation of the subcortical white matter,” Exp. Brain Research, 1999, vol. 125, pp. 383-388. |
| Kitayama, et al. “Regulation of neuronal differentiation by N-methyl-D-aspartate receptors expressed in neural progenitor cells isolated from adult mouse hippocampus,” Journal of Neurosci Research, 2004, vol. 76, No. 5: pp. 599-612. |
| Klausberger, et al. “Brain-state- and cell-type-specific firing of hippocampal interneurons in vivo”, Nature, 2003, vol. 421: pp. 844-848. |
| Kocsis et al., “Regenerating Mammalian Nerve Fibres: Changes in Action Potential Wavefrom and Firing Characteristics Following Blockage of Potassium Conductance”, 1982, Proc. R. Soc. Lond., vol. B 217: pp. 77-87. |
| Knopfel, et al. “Optical Probin of Neuronal Circuit Dynamics: Gentically Encoded Versus Classical Fluorescent Sensors”, 2006, Trends Neurosci, vol. 29, No. 3, pp. 160-166. |
| Kuhlman et al. (2008) “High-Resolution Labeling and Functional Manipulation of Specific Neuron Types in Mouse Brain by Cre-Activated Viral Gene Expression” PLoS One, 2005, vol. 3, No. 4, pp. 1-11. |
| Kunkler, P. et at. “Optical Current Source Density Analysis in Hippocampal Organotypic Culture Shows that Spreading Depression Occurs with Uniquely Reversing Current,” The Journal of Neuroscience, 2005, vol. 25, No. 15, pp. 3952-3961. |
| Landy, A. “Mechanistic and structural complexity in the site-specific recombination pathways of Int and FLP”, Current Opinion in Genetics and Development, 1993, vol. 3, pp. 699-707. |
| Lee et al. “Sterotactic Injection of Adenoviral Vectors that Target Gene Expression to Specific Pituitary Cell Types: Implications for Gene Therapy”, Neurosurgery, 2000, vol. 46, No. 6: pp. 1461-1469. |
| Lee et al., “Potassium Channel Gone Therapy Can Prevent Neuron Deatch Resulting from Necrotic and Apoptotic Insults”, Journal of Neurochemistry, 2003, vol. 85: pp. 1079-1088. |
| Levitan et al. “Surface Expression of Kv1 Voltage-Gated K+ Channels Is Governed by a C-terminal Motif,” Trends Cardiovasc. Med., 2000, vol. 10, No. 7, pp. 317-320. |
| Li et al. “Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.” PNAS, 2005, vol. 102, No. 49, p. 17816-17821. |
| Lim et al., “A Novel Targeting Signal for Proximal Clustering of the Kv2.1K+ Channel in Hippocampal Neurons”, Neuron, 2000, vol. 25: pp. 385-397. |
| Lima, et al. “Remote Control of Behavior through Genetically Targeted Photostimulation of Neurons”, Cell, 2005, vol. 121: pp. 141-152. |
| Liman, et al. “Subunit Stoichiometry of a Mammalian K+ Channel Determined by Construction of Multimeric cDNAs,” Neuron, 1992, vol. 9, pp. 861-871. |
| Louis et al. “Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line,” Virology, 1997, vol. 233, pp. 423-429. |
| Luecke, et al. “Structural Changes in Bacteriorhodopsin During Ion Transport at 2 Angstrom Resolution,” Science, 1999, vol. 286, pp. 255-260. |
| Lyznik, et al. “FLP-mediated recombination of FRT sites in the maize genome,” Nucleic Acids Research, 1996, vol. 24, No. 19: pp. 3784-3789. |
| Ma et al. “Role of ER Export Signals in Controlling Surface Potassium Channel Numbers,” Science, 2001, vol. 291, pp. 316-319. |
| Mann et at. “Perisomatic Feedback Inhibition Underlies Cholinergically Induced Fast Network Oscillations in the Rat Hippocampus in Vitro,” Neuron, 2005, vol. 45, 2005, pp. 105-117. |
| Mattson, “Apoptosis in Neurodegenerative Disorders”, Nature Reviews, 2000, vol. 1: pp. 120-129. |
| Mayberg et al. “Deep Brain Stimulation for Treatment-Resistant Depression,” Focus, 2008, vol. VI, No. 1, pp. 143-154. |
| McAllister, “Cellular and Molecular Mechanisms of Dendrite Growth”, 2000, Cereb Cortex, vol. 10, No. 10, pp. 963-973. |
| McKnight “Functional relationships between transcriptional control signals of the thymidine kinase gene of herpes simplex virus”, Cell, 1982, vol. 31 pp. 355-365. |
| Melyan, Z., et al. “Addition of human melanopsin renders mammalian cells Photoresponsive”, Nature, 2005, vol. 433: pp. 741-745. |
| Mermelstein, et al. “Critical Dependence of cAMP Response Element-Binding Protein Phosphorylation on L-Type Calcium Channels Supports a Selective Response to EPSPs in Preference to Action Potentials”, The Journal of Neuroscience, 2000, vol. 20, No. 1, pp. 266-273. |
| Meyer, et al. “High density interconnects and flexible hybrid assemblies for active biomedical implants,” IEEE Transactions on Advanced Packaging, 2001, vol. 24, No. 3, pp. 366-372. |
| Monje et al., “Irradiation Induces Neural Precursor-Cell Dysfunction”, Natural Medicine, 2002, vol. 8, No. 9, pp. 955-962. |
| Mortensen et al. “Selection of Transfected Mammalian Cells,” Supplement 86, Current Protocols in Molecular Biology, 1997, 9.5.1-09.5.19. |
| Nacher, et al. “NMDA receptor antagonist treatment increases the production of newneurons in the aged rat hippocampus”, Neurobiology of Aging, 2003,vol. 24, No. 2: pp. 273-284. |
| Nagel et al. “Functional Expression of Bacteriorhodopsin in Oocytes Allows Direct Measurement of Voltage Dependence of Light Induced H+ Pumping,” FEBS Letters, 1995, vol. 377, pp. 263-266. |
| Nagel, et al. “Channelrhodopsin-I: a light-gated proton channel in green algae”, Science, 2002, vol. 296: pp. 2395-2398. |
| Nagel, et al. “Channelrhodopsin-2, a directly light-gated cation-selective membrane channel”, PNAS, 2003, vol. 100, No. 24: pp. 13940-13945. |
| Nakagami, et al. “Optical Recording of Trisynaptic Pathway in Rat Hippocampal Slices with a Voltage-Sensitive Dye” Neuroscience, 1997, vol. 81, No. 1, pp. 1-8. |
| Naqvi, et al. “Damage to the insula disrupts addiction to cigarette smoking,” Science; 2007, vol. 315 pp. 531-534. |
| Natochin, et al. “Probing rhodopsin-transducin interaction using Drosophila Rh1-bovine rhodopsin chimeras,” Vision Res., 2006, vol. 46, No. 27: pp. 4575-4581. |
| Nirenberg, et al. “The Light Response of Retinal Ganglion Cells is Truncated by a Displaced Amacrine Circuit”, Neuron, 1997, vol. 18: pp. 637-650. |
| Nunes-Duby, et al. “Similarities and differences among 105 members of the Int family of site-specific recombinases”, Nucleic Acids Research, 1998, vol. 26, No. 2: pp. 391-406. |
| O'Gorman et al. “Recombinase-mediated gene activation and site-specific integration in mammalian cells”, Science, 1991, 251(4999): pp. 1351-1355. |
| Olivares (2001) “Phage R4 integrase mediates site-specific integration in human cells”, Gene, 2001, vol. 278, pp. 167-176. |
| Ory, et al. “A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes,” PNAS, 1996, vol. 93: pp. 11400-11406. |
| Palmer et al., “The Adult Rat Hippocampus Contains Primordial Neural Stem Cells”, Molecular and Cellular Neuroscience, 1997, vol. 8, pp. 389-404. |
| Palmer et al., “Fibroblast Growth Factor-2 Activates a Latent Neurogenic Program in Neural Stem Cells from Diverse Regions of the Adult CNS”, The Journal of Neuroscience, 1999, vol. 19, pp. 8487-8497. |
| Pan et al. “Functional Expression of a Directly Light-Gated Membrane Channel in Mammalian Retinal Neurons: A Potential Strategy for Restoring Light Sensitivity to the Retina After Photoreceptor Degeneration,”Investigative Opthalmology & Visual Science, 2005, 46 E-Abstract 4631. Abstract only. |
| Panda, et al. “Illumination of the Melanopsin Signaling Pathway”, Science, 2005, vol. 307: pp. 600-604. |
| Pape, et al., “Plastic Synaptic Networks of the Amygdala for the Acquisition, Expression, and Extinction of Conditioned Fear”, 2010, Physiol Rev, vol. 90, pp. 419-463. |
| Paulhe et al. “Specific Endoplasmic Reticulum Export Signal Drives Transport of Stem Cell Factor (KitI) to the Cell Surface,” The Journal of Biological Chemistry, 2004, vol. 279, No. 53, p. 55545-55555. |
| Pear “Transient Transfection Methods for Preparation of High-Titer Retroviral Supernatants” Supplement 68, Current Protocols in Molecular Biology, 1996, 9.11.I-9.11.I8. |
| Peterlin, et al. “Optical probing of neuronal circuits with calcium indicators,” PNAS, 2000, vol. 97, No. 7: pp. 3619-3624. |
| Petersen et al. “Spatiotemporal Dynamics of Sensory Responses in Layer 2/3 of Rat Barrel Cortex Measured In Vivo by Voltage-Sensitive Dye Imaging Combined with Whole-Cell Voltage Recordings and Neuron Reconstructions,” The Journal of Neuroscience, 2003, vol. 23, No. 3, pp. 1298-1309. |
| Petrecca, et al. “Localization and Enhanced Current Density of the Kv4.2 Potassium Channel by Interaction with the Actin-Binding Protein Filamin,” The Journal of Neuroscience, 2000, vol. 20, No. 23, pp. 8736-8744. |
| Pettit, et al. “Local Excitatory Circuits in the Intermediate Gray Layer of the Superior Colliculus”, J Neurophysiol., 1999, vol. 81, No. 3: pp. 1424-1427. |
| Potter, “Transfection by Electroporation.” Supplement 62, Current Protocols in Molecular Biology, 1996, 9.3.1-9.3.6. |
| Pouille, et al. “Routing of spike series by dynamic circuits in the hippocampus”, Nature, 2004, vol. 429: pp. 717-723. |
| Qiu et al. “Induction of photosensitivity by heterologous expression of melanopsin”, Nature, 2005, vol. 433: pp. 745-749. |
| Rammes, et al., “Synaptic Plasticity in the Basolateral Amygdala in Transgenic Mice Expressing Dominant-Negative cAMP Response Element-binding Protein (CREB) in Forebrain”, Eur J. Neurosci, 2000, vol. 12, No. 7, pp. 2534-2546. |
| Randic, et al. “Long-term Potentiation and Long-term Depression of Primary Afferent Neurotransmission in the Rat Spinal Cord”, 1993, Journal of Neuroscience, vol. 13, No. 12, pp. 5228-5241. |
| Rathnasingham et al., “Characterization of implantable microfabricated fluid delivery devices,” IEEE Transactions on Biomedical Engineering, 2004, vol. 51, No. 1: pp. 138-145. |
| Ritter, et al., “Monitoring Light-induced Structural Changes of Channelrhodopsin-2 by UV-Visible and Fourier Transform Infared Spectroscopy”, 2008, The Journal of Biological Chemistry, vol. 283, No. 50, pp. 35033-35041. |
| Rivera et al., “BDNF-Induced TrkB Activation Down-Regulates the K+-CI-cotransporter KCC2 and Impairs Neuronal CI-Extrusion”, The Journal of Cell Biology, 2002, vol. 159: pp. 747-752. |
| Rosenkranz, et al. “The prefrontal cortex regulates lateral amygdala neuronal plasticity and responses to previously conditioned stimuli”, J. Neurosci., 2003, vol. 23, No. 35: pp. 11054-11064. |
| Rousche, et al., “Flexible polyimide-based intracortical electrode arrays with bioactive capability,” IEEE Transactions on Biomedical Engineering, 2001, vol. 48, No. 3, pp. 361-371. |
| Rubinson et at. “A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference,” Nature Genetics, 2003, vol. 33, p. 401-406. |
| Rudiger et at. “Specific arginine and threonine residues control anion binding and transport in the light-driven chloride pump halorhodopsin,” The EMBO Journal, 1997, vol. 16, No. 13, pp. 3813-3821. |
| Salzman, et al. “Cortical microstimulation influences perceptual judgements of motion direction”, Nature, 1990, vol. 346, pp. 174-177. |
| Sajdyk, et al., “Excitatory Amino Acid Receptors in the Basolateral Amygdala Regulate Anxiety Responses in the Social Interaction Test”, Brain Research, 1997, vol. 764, pp. 262-264. |
| Sato et al. “Role of Anion-binding Sites in cytoplasmic and extracellular channels of Natronomonas pharaonis halorhodopsin,” Biochemistry, 2005. vol. 44, pp. 4775-4784. |
| Sauer “Site-specific recombination: developments and applications,” Current Opinion in Biotechnology, 1994, vol. 5, No. 5: pp. 521-527. |
| Schiff, et al. “Behavioral improvements with thalamic stimulation after severe traumatic brain injury,” Nature, 2007, vol. 448, pp. 600-604. |
| Schlaepfer et al. “Deep Brain stimulation to Reward Circuitry Alleviates Anhedonia in Refractory Major Depresion,” Neuropsychopharmacology, 2008, vol. 33, pp. 368-377. |
| Sclimenti, et al. “Directed evolution of a recombinase for improved genomic integration at a native human sequence,” Nucleic Acids Research, 2001, vol. 29, No. 24: pp. 5044-5051. |
| Shepherd, et al. “Circuit Analysis of Experience-Dependent Plasticity in the Developing Rat Barrel Cortex”, Neuron, 2003, vol. 38: pp. 277-289. |
| Shibasaki et al. “Effects of body temperature on neural activity in the hippocampus: Regulation of resting membrane potentials by transient receptor potential vanilloid 4,” The Journal of Neuroscience, 2007, vol. 27, No. 7: pp. 1566-1575. |
| Silver, et al. “Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization” PNAS, 1984, vol. 81, No. 19: pp. 5951-5955. |
| Singer et al. “Elevated Intrasynaptic Dopamine Release in Tourette's Syndrome Measured by PET,” American Journal of Psychiatry, 2002, vol. 159: pp. 1329-1336. |
| Slimko et al., “Selective Electrical Silencing of Mammalian Neurons In Vitro by the use of Invertebrate Ligand-Gated Chloride Channels”, The Journal of Neuroscience, 2002, vol. 22, No. 17: pp. 7373-7379. |
| Smith et al. “Diversity in the serine recombinases”, Molecular Microbiology, 2002, vol. 44, No. 2: pp. 299-307. |
| Song et al. “Differential Effect of TEA on Long-Term Synaptic Modification in Hippocampal CA1 and Dentate Gyrus in vitro.” Neurobiology of Learning and Memory, 2001, vol. 76, No. 3, pp. 375-387. |
| Song, “Genes responsible for native depolarization-activated K+ currents in neurons,” Neuroscience Research, 2002, vol. 42, pp. 7-14. |
| Stark, et al. “Catalysis by site-specific recombinases,” Trends Genet., 1992, vol. 8, No. 12: pp. 432-439. |
| Stockklausner et al. “A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K+ channels,” FEBS Letters, 2001, vol. 493, pp. 129-133. |
| Stoll, et al. “Phage TP901-I site-specific integrase functions in human cells,” Journal of Bacteriology, 2002, vol. 184, No. 13: pp. 3657-3663. |
| Swanson, “Lights, Opsins, Action! Optogenetics Brings Complex Neuronal Circuits into Sharper Focus”, 2009, The Dana Foundation, [URL: http://www.dana.org/news/features/detail.aspx?id=24236], PDF File, pp. 1-3. |
| Swiss-Prot—Q2QCJ4, Opsin 1, Oct. 31, 2006, URL: http://www.ncbi.nlm.nig.gov/protein/Q2QCJ4. |
| Takahashi, et al.“Diversion of the Sign of Phototaxis in a Chlamydomonas reinhardtii Mutant Incorporated with Retinal and Its Analogs,” FEBS Letters, 1992, vol. 314, No. 3, pp. 275-279. |
| Tatarkiewicz, et al. “Reversal of Hyperglycemia in Mice After Subcutaneous Transplantation of Macroencapsulated Islets”, Transplantation, 1999, vol. 67, No. 5: pp. 665-671. |
| Tottene et al., “Familial Hemiplegic Migraine Mutations Increase Ca2+ Influx Through Single Human Cav2.1 Current Density in Neurons”, PNAS USA, 2002, vol. 99, No. 20: pp. 13284-13289. |
| Tsau et al. “Distributed Aspects of the Response to Siphon Touch in Aplysia: Spread of Stimulus Information and Cross-Correlation Analysis,” The Journal of Neuroscience, 1994, vol. 14, No. 7, pp. 4167-4184. |
| [No Authors Listed] “Two bright new faces in gene therapy,” Nature Biotechnology, 1996, vol. 14: p. 556. |
| Tye et. al., “Amygdala circuitry mediating reversible and bidirectional control of anxiety”, Nature, 2011, vol. 471(7338): pp. 358-362. |
| Tye et. al., Supplementary Materials: “An optically-resolved microcircuit for bidirectional anxiety control”, Nature, 2011, vol. 471(7338): pp. 358-362. |
| Tye, et al. “Optogenetic investigation of neural circuits underlying brain disease in animal models,” Nature Reviews Neuroscience (Mar. 2012), 13(4):251-266. |
| “SubName: Full=Channelrhodopsin-1”, retrieved from EBI accession No. UNIPROT: B4Y103. Database accession No. B4Y103. Sep. 23, 2008. |
| Ulmanen, et al. “Transcription and translation of foreign genes in Bacillus subtilis by the aid of a secretion vector,” Journal of Bacteriology, 1985, vol. 162, No. 1: pp. 176-182. |
| Van Der Linden, “Functional brain imaging and pharmacotherapy in social phobia: single photon emission computed tomography before and after Treatment with the selective serotonin reuptake inhibitor citalopram,” Prog Neuro-psychopharmacol Biol Psychiatry, 2000, vol. 24, No. 3: pp. 419-438. |
| Vanin, et al. “Development of high-titer retroviral producer cell lines by using Cre-mediated recombination,” Journal of Virology, 1997, vol. 71, No. 10: pp. 7820-7826. |
| Vetter, et al. “Development of a Microscale Implantable Neural Interface (MINI) Probe System,” Proceedings of the 2005 IEEE, Engineering in Medicine and Biology 27th Annual Conference, Shanghai, China, Sep. 1-4, 2005. |
| Wagner, “Noninvasive Human Brain Stimulation”, Annual Rev. Biomed. Eng. 2007. 9:I9.I-19.39. |
| Ward, et al. “Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator”, 1986, Mol. Gen. Genet., vol. 203: pp. 468-478. |
| Watson, et al. “Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins,” Molecular Therapy, 2002, vol. 5, No. 5, pp. 528-537. |
| Wang et al. “Direct-current Nanogenerator Driven by Ultrasonic Waves,” Science, 2007, vol. 316, pp. 102-105. |
| Wang et. al., “High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice”, PNAS, 2007, vol. 104, No. 19, pp. 8143-8148. |
| Weick et al. “Interactions with PDZ Proteins Are Required for L-Type Calcium Channels to Activate cAMP Response Element-Binding Protein-Dependent Gene Expression,” The Journal of Neuroscience, 2003, vol. 23, No. 8, pp. 3446-3456. |
| Wells et al. “Application of Infrared light for in vivo neural stimulation,” Journal of Biomedical Optics, 2005, vol. 10(6), pp. 064003-1-064003-12. |
| Witten et. al., Supporting Online Material for: “Cholinergic Interneurons Control Local Circuit Activity and Cocaine Conditioning”, Science, 2010, vol. 330: 17 pages. |
| Witten et. al., “Cholinergic Interneurons Control Local Circuit Activity and Cocaine Conditioning”, Science, 2010, vol. 330, No. 6011: pp. 1677-1681. |
| Yamazoe, et al. “Efficient generation of dopaminergic neurons from mouse embryonic stem cells enclosed in hollow fibers”, Biomaterials, 2006, vol. 27, pp. 4871-4880. |
| Yan et al., “Cloning and Characterization of a Human β, β-Carotene-15, 15′-Dioxygenase that is Highly Expressed in the Retinal Pigment Epithelium”, Genomics, 2001, vol. 72: pp. 193-202. |
| Yizhar et. al., “Neocortical excitation/inhibition balance in information processing and social dysfunction”, Nature, 2011, vol. 477, pp. 171-178; and Supplemental Materials; 41 pages. |
| Yoon, et al., “A micromachined silicon depth probe for multichannel neural recording,” IEEE Transactions Biomedical Engineering, 2000, vol. 47, No. 8, pp. 1082-1087. |
| Yoshimura, et al. “Excitatory cortical neurons form fine-scale functional networks”, Nature, 2005, vol. 433: pp. 868-873. |
| Zacharias et al. “Recent advances in technology for measuring and manipulating cell signals,” Current Opinion in Neurobiology, 2000, vol. 10: pp. 416-421. |
| Zemelman, et al. “Selective Photostimulation of Genetically ChARGed Neurons”, Neuron, 2002, vol. 33: pp. 15-22. |
| Zemelman, et al. “Photochemical gating of heterologous ion channels: Remote control over genetically designated populations of neurons”, PNAS, 2003, vol. 100, No. 3: pp. 1352-1357. |
| Zhang, et al. “Channelrhodopsin-2 and optical control of excitable cells,” Nature Methods, 2006, vol. 3, No. 10, pp. 785-792. |
| Zhang, et al. “Red-Shifted Optogenetic Excitation: a Tool for Fast Neural Control Derived from Volvox carteri”, Nature Neurosciences, 2008, vol. 11, No. 6, pp. 631-633. |
| Zhang “Multimodal fast optical interrogation of neural circuitry,” Nature, 2007, vol. 446, pp. 633-641. |
| Zrenner, E., “Will Retinal Implants Restore Vision?” Science, 2002, vol. 295, No. 5557, pp. 1022-1025. |
| Zufferey, et al. “Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery”, Journal of Virology, 1998, vol. 72, No. 12, pp. 9873-9880. |
| Adamantidis, et al., “Optogenetic Interrogation of Dopaminergic Modulation of the Multiple Phases of Reward-Seeking Behavior”, J. Neurosci, 2011, vol. 31, No. 30, pp. 10829-10835. |
| Han, et al., “Multiple-Color Optical Activation, Silencing, and Desynchronization of Neural Activity with Single-Spike Temporal Resolution”, PLoS One, 2007, vol. 2, No. 3, pp. 1-12. |
| Kinoshita, et al., “Optogenetically Induced Supression of Neural Activity in the Macaque Motor Cortex”, Poster Sessions Somatomotor System, Others,2010, pp. 141-154. |
| Rein, et al., “The Optogenetic (r)evolution”, Mol. Genet. Genomics, 2012, vol. 287, No. 2, pp. 95-109. |
| Remy, et al., “Depression in Parkinson's Disease: Loss of Dopamine and Noradrenaline Innervation in the Limbic System”, Brain, 2005, vol. 128 (Pt 6), pp. 1314-1322. |
| Tsai, et al., “Phasic Firing in Dopaminergic Neurons in Sufficient for Behavioral Conditioning”, Science, 2009, vol. 324, pp. 1080-1084. |
| Zhao, et al., “Improved Expression of Halorhodopsin for Light-Induced Silencing of Neuronal Activity”, Brain Cell Biology, 2008, vol. 36 (1-4), pp. 141-154. |
| Lanyi et al. “The primary structure of a Halorhodopsin from Natronobacterium pharaonis” Journal of Biological Chemistry 1990, vol. 265, No. 3, p. 1253-1260. |
| Hofherr et al. “Selective Golgi export of Kir2.1 controls the stoichiometry of functional Kir2.x channel heteromers”Journal of Cell Science, 2005, vol. 118, p. 1935-1943. |
| Loetterle, et al., “Cerebellar Stimulation: Pacing the Brain”, American Journal of Nursing, 1975, vol. 75, No. 6, pp. 958-960. |
| Takahashi, et al., “Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors”, 2006, Cell, vol. 126, pp. 663-676. |
| Balint, et al., “The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin”, Biophysical Journal, 2004, vol. 86, pp. 1655-1663. |
| Fiala et al., “Optogenetic approaches in neuroscience”, Current Biology, Oct. 2010, 20(20): R897-R903. |
| Gradinaru et al., “Optical deconstruction of parkinsonian neural circuitry”, Science, Apr. 2009, 324(5925):354-359. |
| Liu et al., “Optogenetics 3.0”, Cell, Apr. 2010, 141(1):22-24. |
| Malin et al., “Involvement of the rostral anterior cingulate cortex in consolidation of inhibitory avoidance memory: Interaction with the basolateral amygdala”, Neurobiol Learning Mem, 2007, 87(2):295-302. |
| Mayford et al., “Control of memory formation through regulated expression of CAMKII Transgene”, Science, Dec. 1996, 274:1678-1683. |
| Schroll et al., “Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae”, Current Biology, Sep. 2006, 16(17):1741-1747. |
| U.S. Appl. No. 13/555,981, filed Jun. 23, 2012, Deisseroth, et al. |
| U.S. Appl. No. 13/622,809, filed Sep. 19, 2012, Deisseroth, et al. |
| U.S. Appl. No. 13/623,612, filed Sep. 20, 2012, Deisseroth, et al. |
| U.S. Appl. No. 13/718,243, filed Dec. 18, 2012, Deisseroth, et al. |
| U.S. Appl. No. 13/763,119, filed Jun. 8, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/763,132, filed Jun. 8, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/772,732, filed Feb. 21, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/847,653, filed Mar. 20, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/847,785, filed Mar. 20, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/849,913, filed Mar. 25, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/850,426, filed Mar. 26, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/850,428, filed Mar. 26, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/850,436, filed Mar. 26, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/850,709, filed Mar. 26, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/854,750, filed Apr. 1, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/854,754, filed Apr. 1, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/855,413, filed Apr. 2, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/875,966, filed May 2, 2013, Deisseroth, et al. |
| U.S. Appl. No. 13/882,566, filed Nov. 4, 2011, Deisseroth, et al. |
| U.S. Appl. No. 13/882,666, filed Nov. 4, 2011, Deisseroth, et al. |
| U.S. Appl. No. 13/882,670, filed Nov. 4, 2011, Deisseroth, et al. |
| U.S. Appl. No. 13/882,703, filed Nov. 4, 2011, Deisseroth, et al. |
| U.S. Appl. No. 13/882,705, filed Nov. 4, 2011, Deisseroth, et al. |
| Tam, B. et al., “Identification of an Outer Segment Targeting Signal in the COOH Terminus of Rhodopsin Using Transgenic Xenopus laevis”, The Journal of Cell Biology, 2000, vol. 151, No. 7, pp. 1369-1380. |
| Gradinaru et al., “Targeting and readout strategies for fast optical neural control in vitro and in vivo”, J Neuroscience, 2007, 27(52):14231-14238. |
| Wang, et al., “Molecular Determinants Differentiating Photocurrent Properties of Two Channelrhodopsins from Chlamydomonas”, 2009, The Journal of Biological Chemistry, vol. 284, No. 9, pp. 5685-5696. |
| Gradinaru, et al., Molecular and Cellular Approaches for Diversifying and Extending Optogenetics, Cell, 2010, vol. 141, No. 1, pp. 154-165. |
| Fox et al., “A gene neuron expression fingerprint of C. elegans embryonic motor neurons”, BMC Genomics, 2005, 6(42):1-23. |
| Nonet, “Visualization of synaptic specializations in live C. elegans with synaptic vesicle protein-GFP fusions”, J. Neurosci. Methods, 1999, 89:33-40. |
| Synapse, Chapter 13, http://michaeldmann.net/mann13.html, downloaded Apr. 2014. |
| RecName: Full=Halorhodopsin; Short=HR; Alt Name: Full=NpHR; XP002704922, retrieved from EBI accession No. UNIPROT: P15647. Database accession No. P15647. Apr. 1, 1990. |
| “Subname: Fluu= Bacteriorhodopsin”; XP002704863, retrieved from EBI accession No. UNIPROT: B0R5N9. Database accession No. B0R5N9. Apr. 8, 2008. |
| “N. pharaonis halorhodopsin (hop) gene, complete cds.”, XP002704883, retrieved from EBI accession No. EMBL: J05199. Database accession No. J05199. Nov. 22, 1990. |
| Zhang, et al., “The Microbial Opsin Family of Optogenetic Tools”, Cell, 2011, vol. 147, No. 7, pp. 1146-1457. |
| Arenkiel, et al. “In vivo light-induced activation of neural circuitry in transgenic mice expressing Channelrhodopsin-2”, Neuron, 2007, 54:205-218. |
| Milella et al. “Opposite roles of dopamine and orexin in quinpirole-induced excessive drinking: a rat model of psychotic polydipsia” Psychopharmacology, 2010, 211:355-366. |
| Marin, et al., The Amino Terminus of the Fourth Cytoplasmic Loop of Rhodopsin Modulates Rhodopsin-Transduction Interaction, The Journal of Biological Chemistry, 2000, vol. 275, pp. 1930-1936. |
| Xiong et al., “Interregional connectivity to primary motor cortex revealed using MRI resting state images”, Hum Brain Mapp, 1999, 8(2-3):151-156. |
| Number | Date | Country | |
|---|---|---|---|
| 20130295015 A1 | Nov 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61464806 | Mar 2011 | US | |
| 61410748 | Nov 2010 | US |
