RESUMEN
Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.
Asunto(s)
Axones , Fibrina , Ganglios Espinales , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Axones/metabolismo , Axones/efectos de los fármacos , Fibrina/metabolismo , Hidrogeles/química , Hidrogeles/farmacología , Ratas , Glucolípidos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/citología , Movimiento Celular/efectos de los fármacosRESUMEN
To investigate the cell behavior underlying neuronal differentiation in a physiologically relevant context, differentiating neurons must be studied in their native tissue environment. Here, we describe an accessible protocol for fluorescent live imaging of differentiating neurons within ex vivo embryonic chicken spinal cord slice cultures, which facilitates long-term observation of individual cells within developing tissue.
Asunto(s)
Diferenciación Celular , Electroporación , Neuronas , Médula Espinal , Animales , Electroporación/métodos , Médula Espinal/citología , Médula Espinal/embriología , Embrión de Pollo , Neuronas/citología , Neuronas/metabolismo , Pollos , NeurogénesisRESUMEN
Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.
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Diferenciación Celular , Interneuronas , Proteínas con Homeodominio LIM , Médula Espinal , Factores de Transcripción , Animales , Proteínas con Homeodominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Interneuronas/metabolismo , Interneuronas/citología , Médula Espinal/citología , Médula Espinal/metabolismo , Médula Espinal/embriología , Embrión de Pollo , Ratones , Neuronas Motoras/metabolismo , Neuronas Motoras/citología , Humanos , Regulación del Desarrollo de la Expresión GénicaRESUMEN
In mammals, the molecular mechanisms underlying transgenerational inheritance of phenotypic traits in serial generations of progeny after ancestral environmental exposures, without variation in DNA sequence, remain elusive. We've recently described transmission of a beneficial trait in rats and mice, in which F0 supplementation of methyl donors, including folic acid, generates enhanced axon regeneration after sharp spinal cord injury in untreated F1 to F3 progeny linked to differential DNA methylation levels in spinal cord tissue. To test whether the transgenerational effect of folic acid is transmitted via the germline, we performed whole-genome methylation sequencing on sperm DNA from F0 mice treated with either folic acid or vehicle control, and their F1, F2, and F3 untreated progeny. Transgenerational differentially methylated regions (DMRs) are observed in each consecutive generation and distinguish folic acid from untreated lineages, predominate outside of CpG islands and in regions of the genome that regulate gene expression, including promoters, and overlap at both the differentially methylated position (DMP) and gene levels. These findings indicate that molecular changes between generations are caused by ancestral folate supplementation. In addition, 29,719 DMPs exhibit serial increases or decreases in DNA methylation levels in successive generations of untreated offspring, correlating with a serial increase in the phenotype across generations, consistent with a 'wash-in' effect. Sibship-specific DMPs annotate to genes that participate in axon- and synapse-related pathways.
Asunto(s)
Axones , Metilación de ADN , Ácido Fólico , Espermatozoides , Ácido Fólico/farmacología , Ácido Fólico/administración & dosificación , Animales , Masculino , Ratones , Espermatozoides/efectos de los fármacos , Espermatozoides/metabolismo , Axones/metabolismo , Axones/efectos de los fármacos , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/metabolismo , Islas de CpG , Femenino , Regeneración Nerviosa/efectos de los fármacos , Epigénesis Genética , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/citologíaRESUMEN
Krause corpuscles, which were discovered in the 1850s, are specialized sensory structures found within the genitalia and other mucocutaneous tissues1-4. The physiological properties and functions of Krause corpuscles have remained unclear since their discovery. Here we report the anatomical and physiological properties of Krause corpuscles of the mouse clitoris and penis and their roles in sexual behaviour. We observed a high density of Krause corpuscles in the clitoris compared with the penis. Using mouse genetic tools, we identified two distinct somatosensory neuron subtypes that innervate Krause corpuscles of both the clitoris and penis and project to a unique sensory terminal region of the spinal cord. In vivo electrophysiology and calcium imaging experiments showed that both Krause corpuscle afferent types are A-fibre rapid-adapting low-threshold mechanoreceptors, optimally tuned to dynamic, light-touch and mechanical vibrations (40-80 Hz) applied to the clitoris or penis. Functionally, selective optogenetic activation of Krause corpuscle afferent terminals evoked penile erection in male mice and vaginal contraction in female mice, while genetic ablation of Krause corpuscles impaired intromission and ejaculation of males and reduced sexual receptivity of females. Thus, Krause corpuscles of the clitoris and penis are highly sensitive mechanical vibration detectors that mediate sexually dimorphic mating behaviours.
Asunto(s)
Clítoris , Mecanorreceptores , Pene , Conducta Sexual Animal , Tacto , Vibración , Animales , Femenino , Masculino , Ratones , Clítoris/inervación , Clítoris/fisiología , Eyaculación/fisiología , Mecanorreceptores/metabolismo , Mecanorreceptores/fisiología , Optogenética , Erección Peniana/fisiología , Pene/inervación , Pene/fisiología , Conducta Sexual Animal/fisiología , Médula Espinal/fisiología , Médula Espinal/citología , Tacto/fisiología , Vagina/fisiología , Neuronas/fisiologíaRESUMEN
During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that ß-arrestin1 and ß-arrestin2 (ß-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that ß-arrestins are expressed in rat commissural neurons. We also found that Smo, ß-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on ß-arrestins. ß-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that ß-arrestins are required to activate Src kinase downstream of Shh. ß-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative ß-arrestins, ß-arrestin1 V53D which blocks the internalization of Smo and ß-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative ß-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that ß-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance.
Asunto(s)
Orientación del Axón , Proteínas Hedgehog , beta-Arrestinas , Animales , Proteínas Hedgehog/metabolismo , Ratas , Orientación del Axón/fisiología , beta-Arrestinas/metabolismo , Arrestinas/metabolismo , Arrestinas/genética , Femenino , Axones/fisiología , Axones/metabolismo , Ratas Sprague-Dawley , Células Cultivadas , Receptor Smoothened/metabolismo , Receptor Smoothened/genética , Familia-src Quinasas/metabolismo , Masculino , Médula Espinal/metabolismo , Médula Espinal/embriología , Médula Espinal/citología , Embrión de Pollo , HumanosRESUMEN
The ventrolateral medulla (VLM) is a crucial region in the brain for visceral and somatic control, serving as a significant source of synaptic input to the spinal cord. Experimental studies have shown that gene expression in individual VLM neurons is predictive of their function. However, the molecular and cellular organization of the VLM has remained uncertain. This study aimed to create a comprehensive dataset of VLM cells using single-cell RNA sequencing in male and female mice. The dataset was enriched with targeted sequencing of spinally-projecting and adrenergic/noradrenergic VLM neurons. Based on differentially expressed genes, the resulting dataset of 114,805 VLM cells identifies 23 subtypes of neurons, excluding those in the inferior olive, and five subtypes of astrocytes. Spinally-projecting neurons were found to be abundant in seven subtypes of neurons, which were validated through in situ hybridization. These subtypes included adrenergic/noradrenergic neurons, serotonergic neurons, and neurons expressing gene markers associated with premotor neurons in the ventromedial medulla. Further analysis of adrenergic/noradrenergic neurons and serotonergic neurons identified nine and six subtypes, respectively, within each class of monoaminergic neurons. Marker genes that identify the neural network responsible for breathing were concentrated in two subtypes of neurons, delineated from each other by markers for excitatory and inhibitory neurons. These datasets are available for public download and for analysis with a user-friendly interface. Collectively, this study provides a fine-scale molecular identification of cells in the VLM, forming the foundation for a better understanding of the VLM's role in vital functions and motor control.
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Bulbo Raquídeo , Neuronas , Médula Espinal , Animales , Bulbo Raquídeo/citología , Bulbo Raquídeo/fisiología , Ratones , Masculino , Femenino , Neuronas/fisiología , Médula Espinal/citología , Médula Espinal/fisiología , Ratones Endogámicos C57BL , Sistema Nervioso Autónomo/fisiología , Sistema Nervioso Autónomo/citologíaRESUMEN
The Hedgehog (HH) pathway is crucial for embryonic development, and adult homeostasis. Its dysregulation is implicated in multiple diseases. Existing cellular models used to study HH signal regulation in mammals do not fully recapitulate the complexity of the pathway. Here we show that Spinal Cord Organoids (SCOs) can be applied to quantitively study the activity of the HH pathway. During SCO formation, the specification of different categories of neural progenitors (NPC) depends on the intensity of the HH signal, mirroring the process that occurs during neural tube development. By assessing the number of NPCs within these distinct subgroups, we are able to categorize and quantify the activation level of the HH pathway. We validate this system by measuring the effects of mutating the HH receptor PTCH1 and the impact of HH agonists and antagonists on NPC specification. SCOs represent an accessible and reliable in-vitro tool to quantify HH signaling and investigate the contribution of genetic and chemical cues in the HH pathway regulation.
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Proteínas Hedgehog , Organoides , Transducción de Señal , Médula Espinal , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Animales , Organoides/metabolismo , Organoides/citología , Médula Espinal/metabolismo , Médula Espinal/citología , Ratones , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Receptor Patched-1/metabolismo , Receptor Patched-1/genéticaRESUMEN
Growing demand for therapeutic tissue repair recurrently focusses scientists' attention on critical assessment of postmortal collection of live cells, especially stem cells. Our study aimed to assess the survival of neuronal progenitors in postmortal spinal cord and their differentiation potential. Postmortal samples of spinal cords were obtained from human-sized animals (goats) at 6, 12, 24, 36, and 54 h after slaughter. Samples were studied by immunohistology, differentiation assay, Western blot and flow cytometry for the presence and location of GD2-positive neural progenitors and their susceptibility to cell death. TUNEL staining of the goat spinal cord samples over 6-54 h postmortem revealed no difference in the number of positive cells per cross-section. Many TUNEL-positive cells were located in the gray commissure around the central canal of the spinal cord; no increase in TUNEL-positive cells was recorded in either posterior or anterior horns of the gray matter where many GD2-positive neural progenitors can be found. The active caspase 3 amount as measured by Western blot at the same intervals was moderately increasing over time. Neuronal cells were enriched by magnetic separation with antibodies against CD24; among them, the GD2-positive neural progenitor subpopulation did not overlap with apoptotic cells having high pan-caspase activity. Apoptotic cell death events are relatively rare in postmortal spinal cords and are not increased in areas of the neural progenitor cell's location, within measured postmortal intervals, or among the CD24/GD2-positive cells. Data from our study suggest postmortal spinal cords as a valuable source for harvesting highly viable allogenic neural progenitor cells.
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Apoptosis , Cabras , Células-Madre Neurales , Médula Espinal , Animales , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Médula Espinal/metabolismo , Médula Espinal/citología , Diferenciación Celular , Supervivencia Celular , Caspasa 3/metabolismoRESUMEN
The development of the vertebrate spinal cord involves the formation of the neural tube and the generation of multiple distinct cell types. The process starts during gastrulation, combining axial elongation with specification of neural cells and the formation of the neuroepithelium. Tissue movements produce the neural tube which is then exposed to signals that provide patterning information to neural progenitors. The intracellular response to these signals, via a gene regulatory network, governs the spatial and temporal differentiation of progenitors into specific cell types, facilitating the assembly of functional neuronal circuits. The interplay between the gene regulatory network, cell movement, and tissue mechanics generates the conserved neural tube pattern observed across species. In this review we offer an overview of the molecular and cellular processes governing the formation and patterning of the neural tube, highlighting how the remarkable complexity and precision of vertebrate nervous system arises. We argue that a multidisciplinary and multiscale understanding of the neural tube development, paired with the study of species-specific strategies, will be crucial to tackle the open questions.
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Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Tubo Neural , Transducción de Señal , Tubo Neural/embriología , Tubo Neural/metabolismo , Tubo Neural/citología , Animales , Tipificación del Cuerpo/genética , Humanos , Redes Reguladoras de Genes , Médula Espinal/embriología , Médula Espinal/citología , Médula Espinal/metabolismo , Diferenciación Celular , Movimiento CelularRESUMEN
Oligodendrocytes, the myelin-producing glial cells of the central nervous system (CNS), crucially contribute to myelination and circuit function. An increasing amount of evidence suggests that intracellular calcium (Ca2+) dynamics in oligodendrocytes mediates activity-dependent and activity-independent myelination. Unraveling how myelinating oligodendrocytes orchestrate and integrate Ca2+ signals, particularly in relation to axonal firing, is crucial for gaining insights into their role in the CNS development and function, both in health and disease. In this framework, we used the recombinant adeno-associated virus/Olig001 capsid variant to express the genetically encoded Ca2+ indicator jGCaMP8s, under the control of the myelin basic protein promoter. In our study, this tool exhibits excellent tropism and selectivity for myelinating and mature oligodendrocytes, and it allows monitoring Ca2+ activity in myelin-forming cells, both in isolated primary cultures and organotypic spinal cord explants. By live imaging of myelin Ca2+ events in oligodendrocytes within organ cultures, we observed a rapid decline in the amplitude and duration of Ca2+ events across different in vitro developmental stages. Active myelin sheath remodeling and growth are modulated at the level of myelin-axon interface through Ca2+ signaling, and, during early myelination in organ cultures, this phase is finely tuned by the firing of axon action potentials. In the later stages of myelination, Ca2+ events in mature oligodendrocytes no longer display such a modulation, underscoring the involvement of complex Ca2+ signaling in CNS myelination.
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Calcio , Dependovirus , Vaina de Mielina , Oligodendroglía , Técnicas de Cultivo de Órganos , Médula Espinal , Animales , Oligodendroglía/metabolismo , Médula Espinal/metabolismo , Médula Espinal/citología , Calcio/metabolismo , Dependovirus/genética , Vaina de Mielina/metabolismo , Señalización del Calcio/fisiología , Ratones Endogámicos C57BL , Ratones , Células Cultivadas , Femenino , RatasRESUMEN
The anterolateral system (ALS) is a major ascending pathway from the spinal cord that projects to multiple brain areas and underlies the perception of pain, itch, and skin temperature. Despite its importance, our understanding of this system has been hampered by the considerable functional and molecular diversity of its constituent cells. Here, we use fluorescence-activated cell sorting to isolate ALS neurons belonging to the Phox2a-lineage for single-nucleus RNA sequencing. We reveal five distinct clusters of ALS neurons (ALS1-5) and document their laminar distribution in the spinal cord using in situ hybridization. We identify three clusters of neurons located predominantly in laminae I-III of the dorsal horn (ALS1-3) and two clusters with cell bodies located in deeper laminae (ALS4 and ALS5). Our findings reveal the transcriptional logic that underlies ALS neuronal diversity in the adult mouse and uncover the molecular identity of two previously identified classes of projection neurons. We also show that these molecular signatures can be used to target groups of ALS neurons using retrograde viral tracing. Overall, our findings provide a valuable resource for studying somatosensory biology and targeting subclasses of ALS neurons.
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Proteínas de Homeodominio , Animales , Ratones , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismo , Neuronas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Masculino , Núcleo Celular/metabolismo , Núcleo Celular/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
A growing body of evidence indicates intra- and inter-regional heterogeneity of astrocytes in the brain. However, because of a lack of an efficient method for isolating astrocytes from the spinal cord, little is known about how much spinal cord astrocytes are heterogeneous in adult mice. In this study, we developed a new method for isolating spinal astrocytes from adult mice using a cold-active protease from Bacillus licheniformis with an astrocyte cell surface antigen-2 (ACSA-2) antibody. Using fluorescence-activated cell sorting, isolated spinal ACSA-2+ cells were divided into two distinct populations, ACSA-2high and ACSA-2low. By analyzing the expression of cell-type marker genes, the ACSA-2high and ACSA-2low populations were identified as astrocytes and ependymal cells, respectively. Furthermore, ACSA-2high cells had mRNAs encoding genes that were abundantly expressed in the gray matter (GM) but not white matter astrocytes. By optimizing enzymatic isolation procedures, the yield of GM astrocytes also increased. Therefore, our newly established method enabled the selective and efficient isolation of GM astrocytes from the spinal cord of adult mice and may be useful for bulk- or single-cell RNA-sequencing under physiological and pathological conditions.
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Astrocitos , Separación Celular , Sustancia Gris , Médula Espinal , Animales , Astrocitos/metabolismo , Astrocitos/citología , Médula Espinal/citología , Separación Celular/métodos , Ratones Endogámicos C57BL , Ratones , Masculino , ARN Mensajero/metabolismo , ARN Mensajero/genética , EnvejecimientoRESUMEN
Resolutive cures for spinal cord injuries (SCIs) are still lacking, due to the complex pathophysiology. One of the most promising regenerative approaches is based on stem cell transplantation to replace lost tissue and promote functional recovery. This approach should be further explored better in vitro and ex vivo for safety and efficacy before proceeding with more expensive and time-consuming animal testing. In this work, we show the establishment of a long-term platform based on mouse spinal cord (SC) organotypic slices transplanted with human neural stem cells to test cellular replacement therapies for SCIs. Standard SC organotypic cultures are maintained for around 2 or 3 weeks in vitro. Here, we describe an optimized protocol for long-term maintenance (≥30 days) for up to 90 days. The medium used for long-term culturing of SC slices was also optimized for transplanting neural stem cells into the organotypic model. Human SC-derived neuroepithelial stem (h-SC-NES) cells carrying a green fluorescent protein (GFP) reporter were transplanted into mouse SC slices. Thirty days after the transplant, cells still show GFP expression and a low apoptotic rate, suggesting that the optimized environment sustained their survival and integration inside the tissue. This protocol represents a robust reference for efficiently testing cell replacement therapies in the SC tissue. This platform will allow researchers to perform an ex vivo pre-screening of different cell transplantation therapies, helping them to choose the most appropriate strategy before proceeding with in vivo experiments.
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Células-Madre Neurales , Traumatismos de la Médula Espinal , Médula Espinal , Animales , Ratones , Traumatismos de la Médula Espinal/terapia , Humanos , Células-Madre Neurales/citología , Células-Madre Neurales/trasplante , Médula Espinal/citología , Técnicas de Cultivo de Órganos/métodos , Trasplante de Células Madre/métodosRESUMEN
Proprioception, the sense of limb and body position, is required to produce accurate and precise movements. Proprioceptive sensory neurons transmit muscle length and tension information to the spinal cord. The function of excitatory neurons in the intermediate spinal cord, which receive this proprioceptive information, remains poorly understood. Using genetic labeling strategies and patch-clamp techniques in acute spinal cord preparations in mice, we set out to uncover how two sets of spinal neurons, Clarke's column (CC) and Atoh1-lineage neurons, respond to electrical activity and how their inputs are organized. Both sets of neurons are located in close proximity in laminae V-VII of the thoracolumbar spinal cord and have been described to receive proprioceptive signals. We find that a majority of CC neurons have a tonic-firing type and express a distinctive hyperpolarization-activated current (Ih). Atoh1-lineage neurons, which cluster into two spatially distinct populations, are mostly a fading-firing type and display similar electrophysiological properties to each other, possibly due to their common developmental lineage. Finally, we find that CC neurons respond to stimulation of lumbar dorsal roots, consistent with prior knowledge that CC neurons receive hindlimb proprioceptive information. In contrast, using a combination of electrical stimulation, optogenetic stimulation, and transsynaptic rabies virus tracing, we find that Atoh1-lineage neurons receive heterogeneous, predominantly local thoracic inputs that include parvalbumin-lineage sensory afferents and local interneuron presynaptic inputs. Altogether, we find that CC and Atoh1-lineage neurons have distinct membrane properties and sensory input organization, representing different subcircuit modes of proprioceptive information processing.
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Propiocepción , Médula Espinal , Animales , Propiocepción/fisiología , Médula Espinal/fisiología , Médula Espinal/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones Transgénicos , Ratones , Masculino , Femenino , Potenciales de Acción/fisiología , Células Receptoras Sensoriales/fisiología , Técnicas de Placa-Clamp , Ratones Endogámicos C57BL , Vértebras TorácicasRESUMEN
The differential expression of transcription factors during embryonic development has been selected as the main feature to define the specific subclasses of spinal interneurons. However, recent studies based on single-cell RNA sequencing and transcriptomic experiments suggest that this approach might not be appropriate in the adult spinal cord, where interneurons show overlapping expression profiles, especially in the ventral region. This constitutes a major challenge for the identification and direct targeting of specific populations that could be involved in locomotor recovery after a traumatic spinal cord injury in adults. Current experimental therapies, including electrical stimulation, training, pharmacological treatments, or cell implantation, that have resulted in improvements in locomotor behavior rely on the modulation of the activity and connectivity of interneurons located in the surroundings of the lesion core for the formation of detour circuits. However, very few publications clarify the specific identity of these cells. In this work, we review the studies where premotor interneurons were able to create new intraspinal circuits after different kinds of traumatic spinal cord injury, highlighting the difficulties encountered by researchers, to classify these populations.
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Interneuronas , Recuperación de la Función , Traumatismos de la Médula Espinal , Adulto , Animales , Humanos , Interneuronas/metabolismo , Médula Espinal/citología , Médula Espinal/patología , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/fisiopatologíaRESUMEN
Neural stem cells have exhibited efficacy in pre-clinical models of spinal cord injury (SCI) and are on a translational path to human testing. We recently reported that neural stem cells must be driven to a spinal cord fate to optimize host axonal regeneration into sites of implantation in the injured spinal cord, where they subsequently form neural relays across the lesion that support significant functional improvement. We also reported methods of deriving and culturing human spinal cord neural stem cells derived from embryonic stem cells that can be sustained over serial high passage numbers in vitro, providing a potentially optimized cell source for human clinical trials. We now report further optimization of methods for deriving and sustaining cultures of human spinal cord neural stem cell lines that result in improved karyotypic stability while retaining anatomical efficacy in vivo. This development improves prospects for safe human translation.
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Diferenciación Celular , Células-Madre Neurales , Traumatismos de la Médula Espinal , Médula Espinal , Humanos , Células-Madre Neurales/citología , Médula Espinal/citología , Animales , Traumatismos de la Médula Espinal/terapia , Diferenciación Celular/fisiología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Ratones , Trasplante de Células Madre/métodosRESUMEN
We are studying the mechanisms of H-reflex operant conditioning, a simple form of learning. Modelling studies in the literature and our previous data suggested that changes in the axon initial segment (AIS) might contribute. To explore this, we used blinded quantitative histological and immunohistochemical methods to study in adult rats the impact of H-reflex conditioning on the AIS of the spinal motoneuron that produces the reflex. Successful, but not unsuccessful, H-reflex up-conditioning was associated with greater AIS length and distance from soma; greater length correlated with greater H-reflex increase. Modelling studies in the literature suggest that these increases may increase motoneuron excitability, supporting the hypothesis that they may contribute to H-reflex increase. Up-conditioning did not affect AIS ankyrin G (AnkG) immunoreactivity (IR), p-p38 protein kinase IR, or GABAergic terminals. Successful, but not unsuccessful, H-reflex down-conditioning was associated with more GABAergic terminals on the AIS, weaker AnkG-IR, and stronger p-p38-IR. More GABAergic terminals and weaker AnkG-IR correlated with greater H-reflex decrease. These changes might potentially contribute to the positive shift in motoneuron firing threshold underlying H-reflex decrease; they are consistent with modelling suggesting that sodium channel change may be responsible. H-reflex down-conditioning did not affect AIS dimensions. This evidence that AIS plasticity is associated with and might contribute to H-reflex conditioning adds to evidence that motor learning involves both spinal and brain plasticity, and both neuronal and synaptic plasticity. AIS properties of spinal motoneurons are likely to reflect the combined influence of all the motor skills that share these motoneurons. KEY POINTS: Neuronal action potentials normally begin in the axon initial segment (AIS). AIS plasticity affects neuronal excitability in development and disease. Whether it does so in learning is unknown. Operant conditioning of a spinal reflex, a simple learning model, changes the rat spinal motoneuron AIS. Successful, but not unsuccessful, H-reflex up-conditioning is associated with greater AIS length and distance from soma. Successful, but not unsuccessful, down-conditioning is associated with more AIS GABAergic terminals, less ankyrin G, and more p-p38 protein kinase. The associations between AIS plasticity and successful H-reflex conditioning are consistent with those between AIS plasticity and functional changes in development and disease, and with those predicted by modelling studies in the literature. Motor learning changes neurons and synapses in spinal cord and brain. Because spinal motoneurons are the final common pathway for behaviour, their AIS properties probably reflect the combined impact of all the behaviours that use these motoneurons.
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Segmento Inicial del Axón , Reflejo H , Neuronas Motoras , Ratas Sprague-Dawley , Animales , Neuronas Motoras/fisiología , Ratas , Masculino , Reflejo H/fisiología , Segmento Inicial del Axón/fisiología , Aprendizaje/fisiología , Médula Espinal/fisiología , Médula Espinal/citología , Axones/fisiología , Plasticidad Neuronal/fisiología , Condicionamiento Operante/fisiología , Ancirinas/metabolismoRESUMEN
The intricate electrophysiological functions and anatomical structures of spinal cord tissue render the establishment of in vitro models for spinal cord-related diseases highly challenging. Currently, both in vivo and in vitro models for spinal cord-related diseases are still underdeveloped, complicating the exploration and development of effective therapeutic drugs or strategies. Organoids cultured from human induced pluripotent stem cells (hiPSCs) hold promise as suitable in vitro models for spinal cord-related diseases. However, the cultivation of spinal cord organoids predominantly relies on Matrigel, a matrix derived from murine sarcoma tissue. Tissue-specific extracellular matrices are key drivers of complex organ development, thus underscoring the urgent need to research safer and more physiologically relevant organoid culture materials. Herein, we have prepared a rat decellularized brain extracellular matrix hydrogel (DBECMH), which supports the formation of hiPSC-derived spinal cord organoids. Compared with Matrigel, organoids cultured in DBECMH exhibited higher expression levels of markers from multiple compartments of the natural spinal cord, facilitating the development and maturation of spinal cord organoid tissues. Our study suggests that DBECMH holds potential to replace Matrigel as the standard culture medium for human spinal cord organoids, thereby advancing the development of spinal cord organoid culture protocols and their application in in vitro modeling of spinal cord-related diseases.
Asunto(s)
Encéfalo , Hidrogeles , Células Madre Pluripotentes Inducidas , Organoides , Médula Espinal , Organoides/efectos de los fármacos , Organoides/citología , Organoides/metabolismo , Humanos , Animales , Médula Espinal/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Hidrogeles/química , Hidrogeles/farmacología , Encéfalo/metabolismo , Ratas , Matriz Extracelular Descelularizada/química , Matriz Extracelular Descelularizada/farmacología , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Laminina/farmacología , Laminina/química , Proteoglicanos/química , Ratas Sprague-Dawley , Combinación de Medicamentos , ColágenoRESUMEN
Spinal cord organoids are of significant value in the research of spinal cord-related diseases by simulating disease states, thereby facilitating the development of novel therapies. However, the complexity of spinal cord structure and physiological functions, along with the lack of human-derived inducing components, presents challenges in the in vitro construction of human spinal cord organoids. Here, we introduce a novel human decellularized placenta-derived extracellular matrix hydrogel (DPECMH) and, combined with a new induction protocol, successfully construct human spinal cord organoids. The human placenta-sourced decellularized extracellular matrix (dECM), verified through hematoxylin and eosin staining, DNA quantification, and immunofluorescence staining, retained essential ECM components such as elastin, fibronectin, type I collagen, laminin, and so forth. The temperature-sensitive hydrogel made from human placenta dECM demonstrated good biocompatibility and promoted the differentiation of human induced pluripotent stem cell (hiPSCs)-derived spinal cord organoids into neurons. It displayed enhanced expression of laminar markers in comparison to Matrigel and showed higher expression of laminar markers compared to Matrigel, accelerating the maturation process of spinal cord organoids and demonstrating its potential as an organoid culture substrate. DPECMH has the potential to replace Matrigel as the standard additive for human spinal cord organoids, thus advancing the development of spinal cord organoid culture protocols and their application in the in vitro modeling of spinal cord-related diseases.