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1.
Neuropsychopharmacology ; 48(12): 1752-1759, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37258714

RESUMO

Early-life stress (ELS) leaves signatures upon the brain that persist throughout the lifespan and increase the risk of psychiatric illnesses including mood and anxiety disorders. In humans, myriad forms of ELS-including childhood abuse, bullying, poverty, and trauma-are increasingly prevalent. Understanding the signs of ELS, including those associated with psychiatric illness, will enable improved treatment and prevention. Here, we developed a novel procedure to model human ELS in mice and identify translationally-relevant biomarkers of mood and anxiety disorders. We exposed male mice (C57BL/6 J) to an early-life (juvenile) chronic social defeat stress (jCSDS) and examined social interaction and responsivity to reward during adulthood. As expected, jCSDS-exposed mice showed a socially avoidant phenotype in open-field social interaction tests. However, sucrose preference tests failed to demonstrate ELS-induced reductions in choice for the sweetened solution, suggesting no effect on reward function. To explore whether other tasks might be more sensitive to changes in motivation, we tested the mice in the Probabilistic Reward Task (PRT), a procedure often used in humans to study reward learning deficits associated with depressive illness. In a touchscreen PRT variant that was reverse-translated to maximize alignment with the version used in human subjects, mice exposed to jCSDS displayed significant reductions in the tendency to develop response biases for the more richly-rewarded stimulus, a hallmark sign of anhedonia when observed in humans. Our findings suggest that translationally-relevant procedures that utilize the same endpoints across species may enable the development of improved model systems that more accurately predict outcomes in humans.


Assuntos
Experiências Adversas da Infância , Humanos , Camundongos , Masculino , Criança , Animais , Adulto , Estresse Psicológico/complicações , Camundongos Endogâmicos C57BL , Encéfalo , Recompensa
2.
Biol Psychiatry ; 94(3): 239-248, 2023 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-36925415

RESUMO

BACKGROUND: The ability to correctly associate cues and contexts with threat is critical for survival, and the inability to do so can result in threat-related disorders such as posttraumatic stress disorder. The prefrontal cortex (PFC) and hippocampus are well known to play critical roles in cued and contextual threat memory processing. However, the circuits that mediate prefrontal-hippocampal modulation of context discrimination during cued threat processing are less understood. Here, we demonstrate the role of a previously unexplored projection from the ventromedial region of PFC (vmPFC) to the lateral entorhinal cortex (LEC) in modulating the gain of behavior in response to contextual information during threat retrieval and encoding. METHODS: We used optogenetics followed by in vivo calcium imaging in male C57/B6J mice to manipulate and monitor vmPFC-LEC activity in response to threat-associated cues in different contexts. We then investigated the inputs to, and outputs from, vmPFC-LEC cells using Rabies tracing and channelrhodopsin-assisted electrophysiology. RESULTS: vmPFC-LEC cells flexibly and bidirectionally shaped behavior during threat expression, shaping sensitivity to contextual information to increase or decrease the gain of behavioral output in response to a threatening or neutral context, respectively. CONCLUSIONS: Glutamatergic vmPFC-LEC cells are key players in behavioral gain control in response to contextual information during threat processing and may provide a future target for intervention in threat-based disorders.


Assuntos
Comportamento , Medo , Vias Neurais , Córtex Olfatório , Córtex Pré-Frontal , Animais , Masculino , Camundongos , Comportamento/fisiologia , Sinalização do Cálcio , Channelrhodopsins/metabolismo , Sinais (Psicologia) , Ácido Glutâmico/metabolismo , Camundongos Endogâmicos C57BL , Córtex Olfatório/citologia , Córtex Olfatório/fisiologia , Optogenética , Córtex Pré-Frontal/citologia , Córtex Pré-Frontal/fisiologia , Transtornos de Estresse Pós-Traumáticos/fisiopatologia , Técnicas de Patch-Clamp
3.
Vet Ophthalmol ; 26 Suppl 1: 16-30, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36786010

RESUMO

Dry eye disease is a complex ophthalmic disorder that consists of two main subtypes, aqueous deficient dry eye (ADDE) and evaporative dry eye disease (EDED). Due to the complex underlying physiology, human dry eye disease can be difficult to model in laboratory animal species. Thus, the identification and characterization of a spontaneous large animal model of dry eye disease is desirable. Dogs have been described as an ideal spontaneous model of ADDE due to the similar pathophysiology between dogs and humans. Recently, EDED and meibomian gland dysfunction (MGD) have been increasingly recognized and reported in dogs. These reports on EDED and MGD in dogs have identified similarities in pathophysiology, clinical presentations, and diagnostic parameters to humans with the comparable disorders. Additionally, the tests that are used to diagnose EDED and MGD in humans are more easily applicable to dogs than to laboratory species due to the comparable globe sizes between dogs and humans. The reported response of dogs to EDED and MGD therapies are similar to humans, suggesting that they would be a valuable preclinical model for the development of additional therapeutics. Further research and clinical awareness of EDED and MGD in dogs would increase their ability to be utilized as a preclinical model, improving the positive predictive value of therapeutics for EDED and MGD in both humans and dogs.


Assuntos
Doenças do Cão , Síndromes do Olho Seco , Disfunção da Glândula Tarsal , Humanos , Cães , Animais , Disfunção da Glândula Tarsal/veterinária , Glândulas Tarsais , Lágrimas , Síndromes do Olho Seco/diagnóstico , Síndromes do Olho Seco/veterinária , Doenças do Cão/diagnóstico
4.
Vet Ophthalmol ; 26 Suppl 1: 125-133, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36478371

RESUMO

OBJECTIVES: This study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies. PROCEDURES: A targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression. RESULTS: Equine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins. CONCLUSIONS: This study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.


Assuntos
Anti-Infecciosos , alfa-Defensinas , beta-Defensinas , Humanos , Animais , Cavalos , beta-Defensinas/genética , beta-Defensinas/metabolismo , alfa-Defensinas/metabolismo , Âmnio/metabolismo , Córnea/metabolismo , Túnica Conjuntiva/metabolismo
5.
Front Microbiol ; 13: 857735, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35722307

RESUMO

Microbial keratitis is a common cause of ocular pain and visual impairment worldwide. The ocular surface has a relatively paucicellular microbial community, mostly found in the conjunctiva, while the cornea would be considered relatively sterile. However, in patients with microbial keratitis, the cornea can be infected with multiple pathogens including Staphylococcus aureus, Pseudomonas aeruginosa, and Fusarium sp. Treatment with topical antimicrobials serves as the standard of care for microbial keratitis, however, due to high rates of pathogen resistance to current antimicrobial medications, alternative therapeutic strategies must be developed. Multiple studies have characterized the expression and activity of antimicrobial peptides (AMPs), endogenous peptides with key antimicrobial and wound healing properties, on the ocular surface. Recent studies and clinical trials provide promise for the use of AMPs as therapeutic agents. This article reviews the repertoire of AMPs expressed at the ocular surface, how expression of these AMPs can be modulated, and the potential for harnessing the AMPs as potential therapeutics for patients with microbial keratitis.

7.
J Equine Vet Sci ; 99: 103394, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33781418

RESUMO

As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in the United States, researchers have investigated the potential for combining equine somatic cell nuclei with oocytes from other species to make embryos for research purposes, which has not been successful to date. There has also been a rising interest in producing transgenic animals using sperm exposed to exogenous DNA. The successful creation of transgenic equine blastocysts shows the promise of sperm mediated gene transfer (SMGT), but this method is not ideal for other applications, like gene therapy, because it cannot be used to induce targeted mutations. That is why technologies like CRISPR/Cas9 are vital. In this review, we argue that parthenogenesis, SCNT, and interspecies SCNT can be considered genetic manipulation strategies as they create embryos that are genetically identical to their parent cell. Here, we describe how these methods are performed and their applications and we also describe the few methods that have been used to directly modify equine embryos: SMGT and CRISPR/Cas9.


Assuntos
Embrião de Mamíferos , Oócitos , Animais , Blastocisto , Cavalos , Masculino , Técnicas de Transferência Nuclear/veterinária , Partenogênese
8.
J Equine Vet Sci ; 98: 103364, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33663726

RESUMO

OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine-specific extrinsic regulators of OCT4 expression that have not yet been investigated. However, an alternative hypothesis is that this restriction may not be evident in equine embryos because of our inability to culture them to the epiblast stage, preventing the observation of this restriction. In vitro studies have identified that OCT4 is expressed in the immature equine oocyte and in the early equine embryo, but OCT4 expression has not been studied after the formation of the ICM in the equine embryo. Despite the gaps in knowledge about equine-specific functions of OCT4, this factor has been used in studies assessing equine embryonic stem cells and to induce pluripotency in equine somatic cells. This review describes the role of OCT4 in the equine embryo and its applications in equine stem cell research.


Assuntos
Células-Tronco Embrionárias , Fator 3 de Transcrição de Octâmero , Animais , Blastocisto , Bovinos , Embrião de Mamíferos , Cavalos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Suínos , Fatores de Transcrição
9.
Elife ; 82019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31829939

RESUMO

Human mutations in the dystroglycan complex (DGC) result in not only muscular dystrophy but also cognitive impairments. However, the molecular architecture critical for the synaptic organization of the DGC in neurons remains elusive. Here, we report Inhibitory Synaptic protein 1 (InSyn1) is a critical component of the DGC whose loss alters the composition of the GABAergic synapses, excitatory/inhibitory balance in vitro and in vivo, and cognitive behavior. Association of InSyn1 with DGC subunits is required for InSyn1 synaptic localization. InSyn1 null neurons also show a significant reduction in DGC and GABA receptor distribution as well as abnormal neuronal network activity. Moreover, InSyn1 null mice exhibit elevated neuronal firing patterns in the hippocampus and deficits in fear conditioning memory. Our results support the dysregulation of the DGC at inhibitory synapses and altered neuronal network activity and specific cognitive tasks via loss of a novel component, InSyn1.


Assuntos
Distroglicanas/metabolismo , Neurônios GABAérgicos/metabolismo , Hipocampo/fisiologia , Memória , Sinapses/metabolismo , Sinapsinas/metabolismo , Animais , Células Cultivadas , Cognição , Humanos , Camundongos Endogâmicos C57BL
10.
Neuron ; 104(3): 559-575.e6, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31447169

RESUMO

Virtuosic motor performance requires the ability to evaluate and modify individual gestures within a complex motor sequence. Where and how the evaluative and premotor circuits operate within the brain to enable such temporally precise learning is poorly understood. Songbirds can learn to modify individual syllables within their complex vocal sequences, providing a system for elucidating the underlying evaluative and premotor circuits. We combined behavioral and optogenetic methods to identify 2 afferents to the ventral tegmental area (VTA) that serve evaluative roles in syllable-specific learning and to establish that downstream cortico-basal ganglia circuits serve a learning role that is only premotor. Furthermore, song performance-contingent optogenetic stimulation of either VTA afferent was sufficient to drive syllable-specific learning, and these learning effects were of opposite valence. Finally, functional, anatomical, and molecular studies support the idea that these evaluative afferents bidirectionally modulate VTA dopamine neurons to enable temporally precise vocal learning.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Aprendizagem/fisiologia , Área Tegmentar Ventral/fisiologia , Vocalização Animal/fisiologia , Animais , Gânglios da Base/fisiologia , Córtex Cerebral/fisiologia , Tentilhões , Masculino , Mesencéfalo/fisiologia , Vias Neurais , Optogenética
11.
Neuron ; 103(4): 583-597.e8, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31272828

RESUMO

Analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, including the diversity of cell types in the brain. However, current approaches are often low throughput and resource intensive. Here, we describe a CRISPR-Cas9-based homology-independent universal genome engineering (HiUGE) method for endogenous protein manipulation that is straightforward, scalable, and highly flexible in terms of genomic target and application. HiUGE employs adeno-associated virus (AAV) vectors of autonomous insertional sequences (payloads) encoding diverse functional modifications that can integrate into virtually any genomic target loci specified by easily assembled gene-specific guide-RNA (GS-gRNA) vectors. We demonstrate that universal HiUGE donors enable rapid alterations of proteins in vitro or in vivo for protein labeling and dynamic visualization, neural-circuit-specific protein modification, subcellular rerouting and sequestration, and truncation-based structure-function analysis. Thus, the "plug-and-play" nature of HiUGE enables high-throughput and modular analysis of mechanisms driving protein functions in cellular neurobiology.


Assuntos
Técnicas de Introdução de Genes/métodos , Genômica/métodos , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Células Cultivadas , Dependovirus/genética , Edição de Genes/métodos , Vetores Genéticos/genética , Humanos , Imunoquímica/métodos , Inteínas , Camundongos , Mutagênese Insercional , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteômica , RNA Guia de Cinetoplastídeos/genética , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência do Ácido Nucleico
13.
Nat Neurosci ; 21(4): 589-597, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29483664

RESUMO

The complex skills underlying verbal and musical expression can be learned without external punishment or reward, indicating their learning is internally guided. The neural mechanisms that mediate internally guided learning are poorly understood, but a circuit comprising dopamine-releasing neurons in the midbrain ventral tegmental area (VTA) and their targets in the basal ganglia are important to externally reinforced learning. Juvenile zebra finches copy a tutor song in a process that is internally guided and, in adulthood, can learn to modify the fundamental frequency (pitch) of a target syllable in response to external reinforcement with white noise. Here we combined intersectional genetic ablation of VTA neurons, reversible blockade of dopamine receptors in the basal ganglia, and singing-triggered optogenetic stimulation of VTA terminals to establish that a common VTA-basal ganglia circuit enables internally guided song copying and externally reinforced syllable pitch learning.


Assuntos
Neurônios Dopaminérgicos/fisiologia , Aprendizagem/fisiologia , Rede Nervosa/fisiologia , Percepção da Altura Sonora/fisiologia , Reforço Psicológico , Área Tegmentar Ventral/citologia , Vocalização Animal/fisiologia , Estimulação Acústica , Fatores Etários , Animais , Gânglios da Base/fisiologia , Channelrhodopsins/genética , Channelrhodopsins/metabolismo , Tentilhões , Masculino , Vias Neurais , Optogenética , Transdução Genética , Tirosina 3-Mono-Oxigenase/metabolismo
14.
Nat Neurosci ; 20(7): 978-986, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28504672

RESUMO

Learning to vocalize depends on the ability to adaptively modify the temporal and spectral features of vocal elements. Neurons that convey motor-related signals to the auditory system are theorized to facilitate vocal learning, but the identity and function of such neurons remain unknown. Here we identify a previously unknown neuron type in the songbird brain that transmits vocal motor signals to the auditory cortex. Genetically ablating these neurons in juveniles disrupted their ability to imitate features of an adult tutor's song. Ablating these neurons in adults had little effect on previously learned songs but interfered with their ability to adaptively modify the duration of vocal elements and largely prevented the degradation of songs' temporal features that is normally caused by deafening. These findings identify a motor to auditory circuit essential to vocal imitation and to the adaptive modification of vocal timing.


Assuntos
Córtex Auditivo/fisiologia , Vias Auditivas/fisiologia , Aprendizagem/fisiologia , Telencéfalo/fisiologia , Vocalização Animal/fisiologia , Envelhecimento/fisiologia , Animais , Animais Geneticamente Modificados , Contagem de Células , Surdez/fisiopatologia , Tentilhões , Masculino , Vias Neurais/fisiologia , Técnicas de Rastreamento Neuroanatômico , Neurônios/fisiologia
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