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1.
Cell Mol Neurobiol ; 44(1): 42, 2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38668880

RESUMO

Lewy Body Dementias (LBD), including Parkinson's disease dementia and Dementia with Lewy Bodies, are characterized by widespread accumulation of intracellular alpha-Synuclein protein deposits in regions beyond the brainstem, including in the cortex. However, the impact of local pathology in the cortex is unknown. To investigate this, we employed viral overexpression of human alpha-Synuclein protein targeting the mouse prefrontal cortex (PFC). We then used in vivo 2-photon microscopy to image awake head-fixed mice via an implanted chronic cranial window to assess the early consequences of alpha-Synuclein overexpression in the weeks following overexpression. We imaged apical tufts of Layer V pyramidal neurons in the PFC of Thy1-YFP transgenic mice at 1-week intervals from 1 to 2 weeks before and 9 weeks following viral overexpression, allowing analysis of dynamic changes in dendritic spines. We found an increase in the relative dendritic spine density following local overexpression of alpha-Synuclein, beginning at 5 weeks post-injection, and persisting for the remainder of the study. We found that alpha-Synuclein overexpression led to an increased percentage and longevity of newly-persistent spines, without significant changes in the total density of newly formed or eliminated spines. A follow-up study utilizing confocal microscopy revealed that the increased spine density is found in cortical cells within the alpha-Synuclein injection site, but negative for alpha-Synuclein phosphorylation at Serine-129, highlighting the potential for effects of dose and local circuits on spine survival. These findings have important implications for the physiological role and early pathological stages of alpha-Synuclein in the cortex.


Assuntos
Espinhas Dendríticas , Camundongos Transgênicos , Córtex Pré-Frontal , alfa-Sinucleína , Animais , Humanos , Masculino , Camundongos , alfa-Sinucleína/metabolismo , Sobrevivência Celular/fisiologia , Espinhas Dendríticas/metabolismo , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Células Piramidais/metabolismo , Células Piramidais/patologia
2.
bioRxiv ; 2023 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-37808820

RESUMO

Lewy Body Dementias (LBD), including Parkinson's disease dementia and Dementia with Lewy Bodies, are characterized by widespread accumulation of intracellular alpha-Synuclein protein deposits in regions beyond the brainstem, including in the cortex. Patients with LBDs develop cognitive changes, including abnormalities in executive function, attention, hallucinations, slowed processing, and cognitive fluctuations. The causes of these non-motor symptoms remain unclear; however, accumulation of alpha-Synuclein aggregates in the cortex and subsequent interference of synaptic and cellular function could contribute to psychiatric and cognitive symptoms. It is unknown how the cortex responds to local pathology in the absence of significant secondary effects of alpha-Synuclein pathology in the brainstem. To investigate this, we employed viral overexpression of human alpha-Synuclein protein targeting the mouse prefrontal cortex (PFC). We then used in vivo 2-photon microscopy to image awake head-fixed mice via an implanted chronic cranial window to assess the early consequences of alpha-Synuclein overexpression in the weeks following overexpression. We imaged apical tufts of Layer V pyramidal neurons in the PFC of Thy1-YFP transgenic mice at 1-week intervals from 1-2 weeks before and 9 weeks following viral overexpression, allowing analysis of dynamic changes in dendritic spines. We found an increase in the relative dendritic spine density following local overexpression of alpha-Synuclein, beginning at 5 weeks post-injection, and persisting for the remainder of the study. We found that alpha-Synuclein overexpression led to an increased percentage and longevity of newly-persistent spines, without significant changes in the total density of newly formed or eliminated spines. A follow up study utilizing confocal microscopy revealed that the increased spine density is found in cortical cells within the alpha-Synuclein injection site, but negative for alpha-Synuclein phosphorylation at Serine-129, highlighting the potential for effects of dose and local circuits on spine survival. These findings have important implications for the physiological role and early pathological stages of alpha-Synuclein in the cortex.

3.
bioRxiv ; 2023 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-37873278

RESUMO

Itch is a protective sensation that drives scratching. Although specific cell types have been proposed to underlie itch, the neural circuit basis for itch remains unclear. Here, we used two-photon Ca2+ imaging of the dorsal horn to visualize the neuronal populations that are activated by itch-inducing agents. We identify a convergent population of spinal neurons that is defined by the expression of GRPR. Moreover, we discover that itch is conveyed to the brain via GRPR-expressing spinal output neurons that target the lateral parabrachial nucleus. Further, we show that nalfurafine, a clinically effective kappa opioid receptor agonist, relieves itch by inhibiting GRPR spinoparabrachial neurons. Finally, we demonstrate that a subset of GRPR spinal neurons show persistent, cell-intrinsic Ca2+ oscillations. These experiments provide the first population-level view of the spinal neurons that respond to pruritic stimuli, pinpoint the output neurons that convey itch to the brain, and identify the cellular target of kappa opioid receptor agonists for the inhibition of itch.

4.
J Biol Chem ; 297(3): 101085, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34411562

RESUMO

The complement cascade is a key component of the innate immune system that is rapidly recruited through a cascade of enzymatic reactions to enable the recognition and clearance of pathogens and promote tissue repair. Despite its well-understood role in immunology, recent studies have highlighted new and unexpected roles of the complement cascade in neuroimmune interaction and in the regulation of neuronal processes during development, aging, and in disease states. Complement signaling is particularly important in directing neuronal responses to tissue injury, neurotrauma, and nerve lesions. Under physiological conditions, complement-dependent changes in neuronal excitability, synaptic strength, and neurite remodeling promote nerve regeneration, tissue repair, and healing. However, in a variety of pathologies, dysregulation of the complement cascade leads to chronic inflammation, persistent pain, and neural dysfunction. This review describes recent advances in our understanding of the multifaceted cross-communication that takes place between the complement system and neurons. In particular, we focus on the molecular and cellular mechanisms through which complement signaling regulates neuronal excitability and synaptic plasticity in the nociceptive pathways involved in pain processing in both health and disease. Finally, we discuss the future of this rapidly growing field and what we believe to be the significant knowledge gaps that need to be addressed.


Assuntos
Via Clássica do Complemento/imunologia , Neuroimunomodulação/fisiologia , Dor Nociceptiva/fisiopatologia , Animais , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Humanos , Imunidade Inata/fisiologia , Neuroimunomodulação/imunologia , Plasticidade Neuronal/fisiologia , Neurônios , Nociceptividade , Dor Nociceptiva/imunologia , Dor/imunologia , Dor/fisiopatologia , Transdução de Sinais
5.
J Neurosci ; 40(46): 8816-8830, 2020 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-33051347

RESUMO

The neurokinin-1 receptor (NK1R; encoded by Tacr1) is expressed in spinal dorsal horn neurons and has been suggested to mediate itch in rodents. However, previous studies relied heavily on neurotoxic ablation of NK1R spinal neurons, which limited further dissection of their function in spinal itch circuitry. To address this limitation, we leveraged a newly developed Tacr1CreER mouse line to characterize the role of NK1R spinal neurons in itch. We show that pharmacological activation of spinal NK1R and chemogenetic activation of Tacr1CreER spinal neurons increases itch behavior in male and female mice, whereas pharmacological inhibition of spinal NK1R suppresses itch behavior. We use fluorescence in situ hybridization (FISH) to characterize the endogenous expression of Tacr1 throughout the superficial and deeper dorsal horn (DDH), as well as the lateral spinal nucleus (LSN), of mouse and human spinal cord. Retrograde labeling studies in mice from the parabrachial nucleus (PBN) show that less than 20% of superficial Tacr1CreER dorsal horn neurons are spinal projection neurons, and thus the majority of Tacr1CreER are local interneurons. We then use a combination of in situ hybridization and ex vivo two-photon Ca2+ imaging of the mouse spinal cord to establish that NK1R and the gastrin-releasing peptide receptor (GRPR) are coexpressed within a subpopulation of excitatory superficial dorsal horn (SDH) neurons. These findings are the first to suggest a role for NK1R interneurons in itch and extend our understanding of the complexities of spinal itch circuitry.SIGNIFICANCE STATEMENT The spinal cord is a critical hub for processing somatosensory input, yet which spinal neurons process itch input and how itch signals are encoded within the spinal cord is not fully understood. We demonstrate neurokinin-1 receptor (NK1R) spinal neurons mediate itch behavior in mice and that the majority of NK1R spinal neurons are local interneurons. These NK1R neurons comprise a subset of gastrin-releasing peptide receptor (GRPR) interneurons and are thus positioned at the center of spinal itch transmission. We show NK1R mRNA expression in human spinal cord, underscoring the translational relevance of our findings in mice. This work is the first to suggest a role for NK1R interneurons in itch and extends our understanding of the complexities of spinal itch circuitry.


Assuntos
Interneurônios , Rede Nervosa/fisiopatologia , Prurido/fisiopatologia , Receptores da Bombesina/biossíntese , Receptores da Bombesina/genética , Receptores da Neurocinina-1/biossíntese , Receptores da Neurocinina-1/genética , Medula Espinal/metabolismo , Medula Espinal/fisiopatologia , Adulto , Animais , Comportamento Animal , Plexo Braquial/fisiopatologia , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Dor/psicologia , Células do Corno Posterior/metabolismo , Prurido/genética , Prurido/psicologia
6.
Pain ; 160(3): 702-711, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30507785

RESUMO

The complement system significantly contributes to the development of inflammatory and neuropathic pain, but the underlying mechanisms are poorly understood. Recently, we identified the signaling pathway responsible for thermal hypersensitivity induced by the complement system component C5a. Here, we examine the mechanisms of another important action of C5a, induction of mechanical hypersensitivity. We found that intraplantar injection of C5a produced a dose-dependent mechanical sensitization and that this effect was blocked by chemogenetic ablation of macrophages in both male and female mice. Knockout of TRPV1 or pretreatment with the TRPV1 antagonists, AMG9810 or 5'-iodoresiniferatoxin (5'-IRTX), significantly reduced C5a-induced mechanical sensitization. Notably, local administration of 5'-IRTX 90 minutes after C5a injection resulted in a slow, but complete, reversal of mechanical sensitization, indicating that TRPV1 activity was required for maintaining C5a-induced mechanical hypersensitivity. This slow reversal suggests that neurogenic inflammation and neuropeptide release may be involved. Indeed, pretreatment with a calcitonin gene-related peptide (CGRP) receptor antagonist (but not an antagonist of the neurokinin 1 receptor) prevented C5a-induced mechanical sensitization. Furthermore, intraplantar injection of CGRP produced significant mechanical sensitization in both wild-type and TRPV1 knockout mice. Taken together, these findings suggest that C5a produces mechanical sensitization by initiating macrophage-to-sensory-neuron signaling cascade that involves activation of TRPV1 and CGRP receptor as critical steps in this process.


Assuntos
Complemento C5a/toxicidade , Hiperalgesia/induzido quimicamente , Hiperalgesia/patologia , Macrófagos/fisiologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Canais de Cátion TRPV/metabolismo , Acrilamidas/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Dipeptídeos/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/metabolismo , Medição da Dor , Piperidinas/farmacologia , Quinazolinas/farmacologia , Quinuclidinas/farmacologia , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Canal de Cátion TRPA1/genética , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPV/genética
7.
Methods Mol Biol ; 1554: 161-173, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28185189

RESUMO

Macrophages are the primary phagocytes of the body and found in every tissue; often with tissue specific subtypes, e.g., microglia or Kupffer Cells. These cells are essential players in host defense, immune regulation, tissue repair, and homeostasis. Consistent with their diverse functions, macrophages display a remarkable level of plasticity and undergo rapid changes in morphology and activation state in response to environmental cues. Polarization of macrophages towards pro-inflammatory (classically activated or M1) or anti-inflammatory (alternatively activated or M2) activation states is highly dependent on their environment. These activation states result in either tissue remodeling and repair (M2) or enhanced inflammation (M1). As macrophages are dependent upon environmental cues for changes in their activation state, primary cell culture offers the ability to study macrophages under highly controlled conditions in which activation states are easily manipulated with specific growth factors, cytokines, or other signaling molecules and are readily examined through powerful tools such as immunostaining, ELISA, and Ca2+ imaging. Additionally, this approach allows the researcher to manipulate gene expression in these cells to better understanding the underlying principles and mechanisms of macrophage biology. Unfortunately, macrophages are resistant to most forms of transfection and researchers have to use either macrophages isolated from transgenic mice or viral delivery of transgenes which slows the study of these diverse cells. In this chapter we describe methods for isolating, culturing, transfecting, and immunostaining primary macrophages. Particular emphasis is placed on culture conditions and transfection protocol as we found these significantly impacted the success of this protocol. Pairing these methods with functional Ca2+ imaging enables investigation of the effects of silencing or overexpressing specific proteins on the functional properties of primary macrophages.


Assuntos
Imuno-Histoquímica , Macrófagos/metabolismo , Cultura Primária de Células , Transfecção , Animais , Biomarcadores , Cálcio/metabolismo , Separação Celular , Eletroporação/métodos , Expressão Gênica , Genes Reporter , Imuno-Histoquímica/métodos , Camundongos , Microscopia de Fluorescência , Imagem Molecular/métodos , Cultura Primária de Células/métodos , RNA Interferente Pequeno/genética , Transfecção/métodos
8.
J Neurosci ; 36(18): 5055-70, 2016 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-27147658

RESUMO

UNLABELLED: The complement cascade is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other components of the complement system in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund's adjuvant was accompanied by C5a upregulation and was markedly reduced by C5a receptor (C5aR1) knock-out or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal hyperalgesia, an effect that was absent in TRPV1 knock-out mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca(2+) mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis mice abolished C5a-dependent thermal hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of NGF, a mediator known to sensitize TRPV1. Preinjection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that complement fragment C5a induces thermal hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF, and TRPV1 as key players in this cross-cellular communication. SIGNIFICANCE STATEMENT: This study provides mechanistic insight into how the complement system, a key component of innate immunity, regulates the development of pain hypersensitivity. We demonstrate a crucial role of the C5a receptor, C5aR1, in the development of inflammatory thermal and mechanical sensitization. By focusing on the mechanisms of C5a-induced thermal hyperalgesia, we show that this process requires recruitment of macrophages and initiation of macrophage-to-nociceptor signaling. At the molecular level, we demonstrate that this signaling depends on NGF and is mediated by the heat-sensitive nociceptive channel TRPV1. This deeper understanding of how immune cells and neurons interact to regulate pain processing is expected to facilitate mechanism-based approaches in the development of new analgesics.


Assuntos
Complemento C5a/metabolismo , Hiperalgesia/fisiopatologia , Macrófagos , Fator de Crescimento Neural , Nociceptores , Transdução de Sinais , Canais de Cátion TRPV , Acrilamidas/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Comunicação Celular , Complemento C5a/genética , Feminino , Temperatura Alta , Inflamação/induzido quimicamente , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Fator de Crescimento Neural/antagonistas & inibidores , Estimulação Física , Canais de Cátion TRPV/antagonistas & inibidores
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