Patent Grant
9968652
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:
Natronomonas
pharaonis
Halobacterium
helobium
Acetabulaira
acetabulum
Guillardia
theta
Leptosphaeria
maculans
Natronomonas
pharaonis
Natronomonas
pharaonis
Table 2 shows identified opsins for excitation and modulation across the visible spectrum:
Volvox
carteri
Dunaliella
sauna
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Chlamydomonas
reinhardtii
Volvox
carteri and
Chlamydomonas
reinhardtii
Volvox
carteri and
Chlamydomonas
reinhardtii
Volvox
carteri and
Chlamydomonas
reinhardtii
Volvox
carteri and
Chlamydomonas
reinhardtii
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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, 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.
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 debilitating1. 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 severity1 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 μm 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 μm).
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-CaMKIIa-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×/0.70 NA or a 40×/1.25 NA 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
μ is the grand mean across all cells (where the ijth “cell” in the collection of observations corresponding to the ith condition and jth treatment)
ci is a fixed effect due to the ith animal condition across treatments (for example, a genetic manipulation)
tj is a fixed effect due to the jth treatment across conditions (for example, light on or light off)
(c:t)ij is a fixed effect due to the interaction of the ith condition and jth treatment in the ijth cell
bj is a random effect corresponding to animals being used across treatments, and
eijk is an independent and identically distributed (i.i.d.) random normal disturbance in the ijkth observation with mean 0 and variance σ2, and independent of bj for all j
Collecting the fixed effects into a 2-way analysis of variance (ANOVA) design matrix XϵRnxp, dummy coding the random effects in a sparse matrix ZϵRnxq, and letting {tilde over (y)}=√{square root over (y)} we can express the model in matrix form as
{tilde over (y)}=Xβ+Zb+e (2)
where {tilde over (y)}ϵn, bϵ
q, and eϵ
n are observations of random variables {tilde over (y)},
, and e respectively and our model assumes
˜
(0,σ2Σ)
ϵ˜(0,σ2I),ϵ⊥
({tilde over (y)}|=b)˜
(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 βϵRp, bϵRq, and the variance parameter σ and sparse (block-diagonal) relative variance-covariance matrix ΣϵRqxq, 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 (
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 is a divisional of U.S. patent application Ser. No. 14/555,048, filed Nov. 26, 2014, now U.S. Pat. No. 9,421,258, which is a divisional of U.S. patent application Ser. No. 13/882,719, filed Jul. 29, 2013, now U.S. Pat. No. 8,932,562, which is a national stage filing under 35 U.S.C. § 371 of PCT/US2011/059298, filed Nov. 4, 2011, which claims the priority benefit of U.S. provisional application Ser. No. 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.
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| Parent | 14555048 | Nov 2014 | US |
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