1997 — 2006 |
Pereda, Alberto E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Control of Junctional Conductance At Auditory Afferents @ Albert Einstein Col of Med Yeshiva Univ
DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to study the role and properties of electrical synaptic transmission via gap junctions in the CNS, in particular in the auditory system. The experimental model involves identified mixed electrical and chemical, (glutamatergic) synapses between eighth nerve auditory primary afferents and the goldfish Mauthner (M-) cell. While most studies of gap junction function utilize exogenous expression systems, this preparation uniquely allows continuous monitoring and quantification of changes in junctional conductance in vivo. Both components of the synaptic response exhibit activity-dependent modifications on their strength that is mediated via activation of NMDA receptors. Paired intradendritic and single afferent recordings, molecular biology techniques, and immunocytochemistry, will be used to test specific hypotheses and mechanisms underlying modifications of electrical transmission induced by eighth nerve tetani, determinants of bi-directional communication and the identity of specific gap junction proteins. Aim 1 explores the cellular and molecular mechanisms underlying activity-dependent modification (potentiation and depression) of gap junctional conductance. It is based on data suggesting that changes in electrical coupling at single terminals following brief tetani can be in the form of both depressions and potentiations. I will explore the roles of elevated levels of postsynaptic calcium/calmodulin-dependent kinase II (CamKIl), protein phosphatases and agents interfering with postsynaptic exofendocytosis on unitary and population synaptic responses. Aim 2 is to investigate the possibIe role of somatostatin in activity-dependent plasticity of these junctions. This peptide is co-localized with glutamate at presynaptic terminals and preliminary data shows that its application enhances both components of the synaptic response. Since both somatostatin and glutamate are likely to be co-released during tetani, I propose to explore their possible functional interactions and underlying intracellular mechanisms. Aim 3 concerns identification of the neuron-specific gap junction proteins at these connections. Sub-cellular distributions of antibodies specific to various connexins will be analyzed with immunocytochemistry, using confocal and freeze-fracture electron microscopy, and single cell RT-PCR of the coupled cells. The proposed research addresses the concept that intercellular coupling through gap junction channels is dynamic, based on its functional interaction with neighboring glutamatergic synapses and peptidergic transmission. These modulatory phenomena could constitute a widespread property of electrical synapses in general, relevant not only to normal brain function in structures such as the retina, inferior olive, and neocortex where both forms of transmission co-exist, but also to numerous health-related issues such as epilepsy.
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0.922 |
2006 |
Pereda, Alberto E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
"in-Vivo Modulation of Synapses by Endocannabinoids" @ Albert Einstein Col of Med Yeshiva Univ
[unreadable] DESCRIPTION (provided by applicant): The long-term objective of the proposed research is to study the modulatory actions of endocannabinoids on synaptic transmission in the CNS. The experimental model involves identified mixed electrical and chemical (glutamatergic) synapses between eighth nerve auditory primary afferents and the goldfish Mauthner (M-) cell and neighboring (GABA/Glycine) inhibitory terminals. While most studies describing the role of endocannabinoids on synaptic transmission have utilized in-vitro systems, this preparation uniquely allows continuous monitoring and quantification of changes in electrical and chemical transmission in-vivo. So far, endocannabinoids have been reported to depress chemical synaptic transmission via presynaptic activation of cannabinoid type 1 receptors (CB1Rs). Contrasting this notion, our preliminary results show that activation of CB1Rs enhances synaptic transmission at these inputs on the M-cell. Intradendritic recordings, molecular biology techniques, and immunocytochemistry, will be used to test specific hypotheses and mechanisms underlying modifications of synaptic transmission induced by this agonist. Aim 1, explores the action of different cannabinoid agonists and endocannabinoids on the synaptic efficacy of mixed synapses and inhibitory terminals. It is based on data suggesting that activation of CB1R leads to long-lasting enhancement of both electrical and chemical transmission at mixed synapses. These changes also included nearby inhibitory terminals. I will explore the actions of locally applied cannabinoid agonists and endocannabinoids on unitary and population synaptic responses and membrane conductances that are relevant for the function of this auditory input. Aim 2 is to investigate the mechanisms underlying these long-term changes in synaptic transmission. It is based on the finding that dopamine receptor antagonists block the potentiation triggered by CB1R activation. We have previously reported the presence of a dopaminergic innervation and application of dopamine evoked lasting enhancements of the synaptic response. We will test the hypothesis that cannabinoid-evoked potentiation is mediated via dopamine release from neighboring varicosities. We will also ask under which physiological conditions and from which particular cell type endocannabinoids are released. The proposed research addresses the concept that modulation of intercellular communication by endocannabinoids is not restricted to chemical synapses but also include gap-junction mediated electrical synapses. Moreover, based on a functional interaction with the dopaminergic system, it can lead to long-term potentiation of synaptic responses. This modulatory action could constitute a widespread property, relevant not only to normal brain function in structures such as the basal ganglia, retina, and neocortex where both forms of transmission co-exist, but also to numerous health-related issues such as drug abuse. [unreadable] [unreadable]
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0.922 |
2007 — 2009 |
Pereda, Alberto E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
In-Vivo Modulation of Synapses by Endocannabinoids @ Albert Einstein College of Medicine
The long-term objective of the proposed research is to study the modulatory actions of endocannabinoids on synaptic transmission in the CNS. The experimental model involves identified mixed electrical and chemical, (glutamatergic) synapses between eighth nerve auditory primary afferents and the goldfish Mauthner (M-) cell and neighboring (GABA/Glycine) inhibitory terminals. While most studies describing the role of endocannabinoids on synaptic transmission have utilized in-vitro systems, this preparation uniquely allows continuous monitoring and quantification of changes in electrical and chemical transmission in-vivo. Sofar, endocannabinoids have been reported to depress chemical synaptic transmission via presynaptic activation of cannabinoid type 1 receptors (CBIRs). Contrasting this notion, our preliminary results show that activation ofCBIRs enhances synaptic transmission at these inputs on the M-cell. Intradendritic recordings, molecular biology techniques, and immunocytochemistry, will be used to test specific hypotheses and mechanisms underlying modifications of synaptic transmission induced by this agonist. Aim 1, explores the action of different cannabinoid agonists and endocannabinoids on the synaptic efficacy of mixed synapses and inhibitory terminals. It is based on data suggesting that activation of CB1R leads to long-lasting enhancement of both electrical and chemical transmission at mixed synapses. These changes also included nearby inhibitory terminals. I will explore the actions of locally applied cannabinoid agonists and endocannabinoids on unitary and population synaptic responses and membrane conductances that are relevant for the function of this auditory input. Aim 2 is to investigate the mechanisms underlying these long-term changes in synaptic transmission. It is based on the finding that dopamine receptor antagonists block the potentiation triggered by CB1R activation. We have previously reported the presence of a dopaminergic innervation and application of dopamine evoked lasting enhancements of the synaptic response. We will test the hypothesis that cannabinoid-evoked potentiation is mediated via dopamine release from neighboring varicosities. We will also ask under which physiological conditions and from which particular cell type endocannabinoids are released. The proposed research addresses the concept that modulation of intercellular communication by endocannabinoids is not restricted to chemical synapses but also include gap-junction mediated electrical synapses. Moreover, based on a functional interaction with the dopaminergic system, it can lead to long-term potentiation of synaptic responses. This modulatory action could constitute a widespread property, relevant not only to normal brain function in structures such as the basal ganglia, retina, and neocortex where both forms of transmission co-exist, but also to numerous health- related issues such as drug abuse.
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0.958 |
2007 — 2008 |
Pereda, Alberto E |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Plasticity of Mammalian Electrical Synapses @ Albert Einstein Col of Med Yeshiva Univ
[unreadable] DESCRIPTION (provided by applicant): The past few years marks a renaissance in the study of electrical synapses which have been shown to exist in an ever-increasing number of areas across the mammalian brain. Despite the overwhelming evidence for their importance and widespread distribution, still little is known about their ability to undergo plastic changes. The notion that mammalian electrical synapses could be as dynamic and modifiable as chemical synapses could dramatically change our perception about their properties and functional relevance. Electrical synapses at identifiable mixed synaptic contacts on goldfish Mauthner cells are regulated by their co-localized glutamatergic synapses, whose activity induces long-term potentiation of electrical transmission via NMDA receptor activation. Recent data show that electrical transmission at these terminals is mediated by connexin35, the fish ortholog of the mammalian neuronal connexin36. The widespread distribution of connexin36 and the ubiquity of the proposed regulatory elements suggest that mammalian electrical synapses may be similarly regulated. We propose to test this prediction at electrical synapses in the rat, in particular at those of the Inferior Olive, where ultrastructural and physiological features appear to favor such possibility. Aim 1 tests the hypothesis that electrical synapses between inferior olivary cells are regulated by the activity of neighboring glutamatergic synapses. It is based on preliminary ultrastructural studies suggesting that, as in goldfish mixed synapses, gap junctions labeled for connexin36 are in close proximity to postsynaptic densities labeled for NMDA receptors, sufficiently close for diffusion of signaling molecules between the two types of structures. Aim 2 is to investigate the mechanisms underlying activity-dependent changes in electrical transmission. We will ask if the mechanistic requirements are similar to those found for Mauthner cell synapses (involving NMDA receptor activation of CaM-KII) or, alternatively, different signaling pathways are involved. The proposed research addresses the novel concept that the strength of mammalian electrical synapses is dynamically modified by the activity of nearby chemical synapses. This property could be widespread and relevant to pathological conditions such as epilepsy and developmental disorders. The application explores the possibility that chemically mediated synapses, the main form of interneuronal communication in the mammalian brain, regulate the function of gap junction-mediated electrical synapses. Because electrical synapses have been shown to promote coordinated neuronal activity, the existence of such regulation could have profound physiological and pathological implications, contributing to epilepsy and to cognitive (psychiatric) and developmental disorders. [unreadable] [unreadable]
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0.922 |
2009 |
Pereda, Alberto E |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Control of Junctional Conductance At Auditory Afferent @ Albert Einstein College of Medicine
Abstract The long-term objective of the proposed research is to study the role and properties of electrical synaptic transmission via gap junctions in the CNS, in particular in the auditory system. The goldfish Mauthner (M-) cell system is ideally suited for these studies since unlike mammalian electrical synapses, the experimental accessibility makes it possible to quantify in vivo changes in junctional conductance that occur under different physiological conditions and to correlate them with anatomical, ultrastructural and molecular analysis. Our progress shows that the conductance of these electrical synapses is under the fine regulatory control of glutamatergic synapses co-localized in the same terminals. Further, our progress also shows that electrical transmission is mediated by Connexin 35 (Cx35), the fish ortholog of the mammalian Connexin 36, suggesting that mammalian electrical synapses could be similarly modulated. This proposal focuses now on understanding the molecular mechanisms underlying changes in junctional conductance. We will investigate if, in analogy to the role of scaffold proteins in chemical synapses, mechanisms of exo/endocytosis involving interactions with the scaffold ZO-1 are necessary for activity-dependent potentiation of junctional conductance. Aim 1, is to investigate the association and interaction of Cx35 with the scaffold protein ZO-1. The scaffold protein ZO-1 is known to interact with many connexins to regulate their surface expression. Our preliminary results indicate that this protein co-localizes and directly interacts with Cx35 through conserved regions of both Cx35 and Cx36 carboxy-terminus. We propose to characterize direct protein-protein interactions between Cx35 and ZO-1. Aim 2, investigates the role of the Cx35/ZO-1 association in regulating electrical synaptic transmission. It is based on evidence suggesting the existence of active trafficking of gap junction channels and that potentiation could be prevented by intracellular injections of both botulinum toxin (that block exocytosis) and by peptides that interfere with Cx35/ZO-1 interactions. The proposed research addresses the novel concept that the strength of electrical synapses is dynamically modified by the activity of nearby chemical synapses. ZO-1 could become as a result of the proposed investigations the first regulatory protein identified for electrical transmission. Furthermore, its direct interaction through conserved regions of both Cx35 and Cx36 carboxy-terminus suggests that its function might underlie a fundamental and widespread property of electrical transmission, also relevant to mammalian electrical synapses. This property could be widespread and relevant to pathological conditions such as epilepsy and developmental disorders.
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0.958 |
2010 — 2014 |
Pereda, Alberto E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Plasticity of Electrical Synapses @ Albert Einstein College of Medicine, Inc
DESCRIPTION (provided by applicant): The goal of this proposal is to investigate the role and properties of gap junction-mediated electrical synapses in the auditory system. Auditory afferents terminating as large mixed (electrical and chemical) synaptic terminals on the goldfish Mauthner cell are ideally suited for these studies since unlike mammalian electrical synapses, the experimental accessibility makes it possible to quantify in vivo changes in junctional conductance that occur under different physiological conditions and to correlate them with anatomical, ultrastructural and molecular analysis. Strikingly, the conductance of these model electrical synapses is under the fine regulatory control of neuronal activity. Electrical transmission is mediated by connexin 35 (Cx35), the fish ortholog of the mammalian connexin 36 (Cx36) which is present in the auditory system, suggesting that mammalian auditory electrical synapses could be similarly regulated. This proposal deals on understanding the molecular mechanisms underlying the bi-directional control of junctional conductance at these terminals by focusing in the role of regulated trafficking of gap junction channels. Aim 1 is to investigate the existence of trafficking of gap junction channels at native electrical synapses, in vivo, by combining ultrastructural and pharmacological approaches. Our preliminary results suggest the existence of an active turnover of gap junction channels, which constitute the first evidence of this phenomenon in a native synapse. Aim 2 is to determine the contribution of regulated trafficking to activity-dependent potentiation of electrical transmission. It will test if mechanisms of exocytosis are required for the expression of the potentiation and if it requires of direct interactions with the regulatory kinase CaM-KII and the scaffold protein ZO-1. Conversely, Aim 3 will investigate the possible contribution of regulated trafficking to activity-dependent depression of electrical transmission. Using similar approaches, we will investigate if mechanisms of endocytosis are required for activity-dependent depression of junctional conductance, as well the potential roles of direct protein-protein interactions. Furthermore, the direct interactions of CaM-KII and ZO-1 through conserved regions of both Cx35 and Cx36 suggests that its function might underlie a fundamental and widespread property of electrical transmission, also relevant to mammalian electrical synapses. Thus, the proposed research addresses the novel concept that the strength of electrical synapses is achieved by dynamically regulating the trafficking of gap junction channels. Because electrical synapses have been shown to promote coordinated neuronal activity, dysfunction of this regulation could have profound pathological implications, contributing to auditory impairment, epilepsy and cognitive (psychiatric) and developmental disorders
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0.958 |
2013 — 2014 |
Pereda, Alberto E |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Generation of Transgenic Zebrafish to Study Electrical Synaptic Transmission @ Albert Einstein College of Medicine, Inc
DESCRIPTION (provided by applicant): Gap junction (GJ) mediated electrical synaptic transmission is considered an essential form of interneuronal communication. It critically contributes to important functional processes in diverse regions of the mammalian CNS and has been linked to a variety of neurological conditions. Plasticity of electrical synapses underlies important functions by reconfiguring networks of electrically coupled neurons, whose disruption might contribute to neurological dysfunction. In contrast to chemical synapses, less is known regarding the molecular mechanisms that regulate the strength of electrical synapses. This proposal focuses on understanding mechanisms underlying plastic changes in GJ communication observed at mixed, electrical and chemical, synapses that couple primary auditory afferents to the teleost Mauthner (M-) cells, at which GJs are formed by fish homologs of the widespread mammalian GJ protein connexin36 (Cx36) and where it is possible to analyze cellular and sub-cellular mechanisms in-vivo. Our studies in goldfish show that both components of the mixed synaptic response undergo activity-dependent potentiation of their respective strengths. Remarkably, our recent findings indicate that factors regulating the turnover and number of functional GJ channels might constitute major determinants of the strength of electrical transmission. We propose here to investigate the contribution of trafficking of GJ channels as a possible mechanism for regulating the strength of electrical transmission. For this purpose, we will take this unique model mixed synapse to a new level of analysis by investigating their properties in larval zebrafish, whose transparency will make it possible to track individual molecules within living cells, in vivo. Supporting this possibility, our preliminay results indicate that mixed synapses in larval zebrafish are molecularly and functionally analogous to those of adult goldfish. The proposal has two aims: Aim 1 is to generate transgenic zebrafish in which neuronal gap junction proteins are tagged with fluorescent proteins, and Aim 2 is to investigate the turnover of fluorescently tagged gap junction channels in-vivo and its properties under conditions that trigger plasticity. The amenability of zebrafish larvae to image the movement of fluorescently tagged GJ channels in-vivo should permit the monitoring of active synapses undergoing plasticity providing an unprecedented window for the analysis of this modality of transmission at which detailed molecular mechanisms could be investigated combining electrophysiology and live imaging with powerful genetic manipulations. Thus, the development of this zebrafish model will provide a new powerful tool to study molecular aspects of Cx36-mediated synapses (prevalent in mammals) that could lead to the identification of novel therapeutic opportunities for the treatment of various neurological conditions.
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0.958 |
2017 — 2021 |
Pereda, Alberto E |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Plasticity of Auditory Electrical Synapses @ Albert Einstein College of Medicine
Abstract Gap junction (GJ)-mediated electrical synapses were recently reported to underlie important network properties in the dorsal cochlear nucleus and anatomical evidence suggests they are widespread along the auditory pathway. However, the properties of auditory electrical synapses remain poorly understood. As their chemical counterparts, electrical synapses are ?plastic?, that is, they modify their strength with activity. Changes in the strength of electrical synapses dynamically reconfigure neuronal circuits in various neural structures. Thus, the presence and plastic properties of electrical synapses could fundamentally change the way we understand the organization of auditory circuits and, ultimately, the processing of auditory information. This proposal aims to contribute to our understanding of electrical transmission in the auditory system by investigating the molecular mechanisms causing plastic changes in GJ communication at mixed, electrical and chemical, contacts that couple primary auditory afferents to the Mauthner (M-) cells in fish. Our work in goldfish shows that electrical (and chemical) transmission at these mixed synapses undergo activity-dependent potentiation. Because these dynamic properties were later found to occur at mammalian electrical synapses. M-cell mixed synapses are considered a valuable model to study plasticity of vertebrate electrical transmission. In contrast to chemical synapses, little is known about the molecular mechanisms that underlie changes in the strength of electrical synapses. It is currently thought that plastic changes in GJ conductance are due to direct modification of the properties of already existing channels. However, our progress suggests that regulated insertion and removal of GJ channels may also contribute to plasticity. We propose to investigate the contribution of regulated trafficking of GJ channels to plastic changes of electrical transmission and its molecular underpinnings. To directly examine this possibility, we will take these unique model mixed synapses to a new level of analysis by investigating their properties in larval zebrafish. The amenability of zebrafish larvae to image the movement of fluorescently-tagged GJ channels in-vivo should allow monitoring of active synapses undergoing plasticity. This approach will provide an unprecedented window for the analysis of electrical transmission at which detailed molecular mechanisms will be investigated by combining in-vivo imaging, electrophysiology and time-resolved ultrastructural analysis with powerful genetic manipulations. Aim 1 is to investigate the conditions under which electrical synapses in larval zebrafish undergo potentiation. By combining electrophysiology and pharmacology with electrical and optogenetic stimulation, this aim will identify the conditions under which larval mixed synapses undergo potentiation of electrical (and chemical) transmission. Aim 2 is to test whether insertion and removal of GJ channels are required for plastic changes. This aim will explore the notion that electrical synapses are complex synaptic structures at which channels turnover and that their proper function and regulation results from interactions between multiple proteins. The description of novel molecular mechanisms involved in their regulation will contribute to a better understanding of the dynamics of circuits relevant to auditory dysfunction and the potential identification of novel therapeutic targets.
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0.958 |
2020 |
Miller, Adam C (co-PI) [⬀] O'brien, John Pereda, Alberto E |
RF1Activity Code Description: To support a discrete, specific, circumscribed project to be performed by the named investigator(s) in an area representing specific interest and competencies based on the mission of the agency, using standard peer review criteria. This is the multi-year funded equivalent of the R01 but can be used also for multi-year funding of other research project grants such as R03, R21 as appropriate. |
Transgenic Tools For Revealing the Contributions of Electrical Synapses to Neural Circuits @ Albert Einstein College of Medicine
Abstract While current efforts in the analysis of neural circuits focus on interneuronal connectivity mediated by chemical synapses, less is known about the contribution of electrical synapses. Electrical transmission is mediated by neuronal gap junctions, which are widely distributed throughout the vertebrate brain. However, the extent and subcellular distribution of electrical synapses within neural circuits has been difficult to assess because: 1) antibodies targeting connexins (gap junction forming proteins) vary in their specificity, resulting in false positive or negative staining, and therefore potentially generating wrong or incomplete maps of connectivity, and 2) current electron microscopy protocols used to generate connectomes are unfavorable for detecting gap junctions, thus biasing the description of neuronal interconnection to chemical synapses. To overcome this problem, we propose to develop transgenic-based methods that will allow investigating the presence and contribution of electrical synapses in zebrafish, a model organism that has been identified as particularly advantageous for the analysis of neural circuits by the Brain Initiative. More specifically, we propose to create a Library of Transgenic Zebrafish to study Electrical Synaptic Transmission which will make it possible to generate, for the first time, a complete map of the distribution of electrical synapses in a vertebrate nervous system. The proposal involves generating three types of fish at which connexins and/or its promoters are tagged with fluorescent proteins or functional sensors that, combined, will allow comprehensive examination of the functional contributions of electrical synapses to circuits underlying various behaviors with cell specificity. Aim 1 is to generate transgenic zebrafish at which the promoters of neuronal connexins are linked to reporter fluorescent proteins. The availability of these animals will allow for the establishment of the presence of a particular gap junction protein in a cell or circuit of interest, a notoriously challenging problem, as cells expressing a particular connexin will be fluorescently labeled. Aim 2 is to generate transgenic zebrafish at which zebrafish neuronal connexins are tagged with fluorescent proteins. We will engineer the endogenous neuronal connexin proteins with fluorescent proteins or affinity tags to assess the number and subcellular location of electrical synapses of a cell with its connected neighbors. Because of the design of the constructs, tagged connexins can be imaged by diverse methods including single or 2-photon imaging of living animals or tissues, or chemical enhancements suitable for electron microscopic analysis. Finally, Aim 3 is to generate transgenic zebrafish to study functional contributions of electrical synapses to neuronal circuits. We propose to generate transgenic fish in which neuronal connexins are linked to Ca++ sensors that will make possible detecting active electrical synapses as well those undergoing plastic changes. The proposed approach represents a significant improvement over current methods of analysis and, if successful for analysis of zebrafish neural circuits, could be potentially applied to analysis of electrical transmission in mammalian species.
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0.958 |