Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

Glial cells maintain synapses by inhibiting an activity-dependent retrograde protease signal.

Glial cells regulate multiple aspects of synaptogenesis. In the absence of Schwann cells, a peripheral glial cell, motor neurons initially innervate muscle but then degenerate. Here, using a genetic approach, we show that neural activity-regulated negative factors produced by muscle drive neurodegeneration in Schwann cell-deficient mice. We find that thrombin, the hepatic serine protease central to the hemostatic coagulation cascade, is one such negative factor. Trancriptomic analysis shows that expression of the antithrombins serpin C1 and D1 is significantly reduced in Schwann cell-deficient mice. In the absence of peripheral neuromuscular activity, neurodegeneration is completely blocked, and expression of prothrombin in muscle is markedly reduced. In the absence of muscle-derived prothrombin, neurodegeneration is also markedly reduced. Together, these results suggest that Schwann cells regulate NMJs by opposing the effects of activity-regulated, muscle-derived negative factors and provide the first genetic evidence that thrombin plays a central role outside of the coagulation system.

Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms.

Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.

Neurotransmitter acetylcholine negatively regulates neuromuscular synapse formation by a Cdk5-dependent mechanism.

Synapse formation requires interactions between pre- and postsynaptic cells to establish the connection of a presynaptic nerve terminal with the neurotransmitter receptor-rich postsynaptic apparatus. At developing vertebrate neuromuscular junctions, acetylcholine receptor (AChR) clusters of nascent postsynaptic apparatus are not apposed by presynaptic nerve terminals. Two opposing activities subsequently promote the formation of synapses: positive signals stabilize the innervated AChR clusters, whereas negative signals disperse those that are not innervated. Although the nerve-derived protein agrin has been suggested to be a positive signal, the negative signals remain elusive. Here, we show that cyclin-dependent kinase 5 (Cdk5) is activated by ACh agonists and is required for the ACh agonist-induced dispersion of the AChR clusters that have not been stabilized by agrin. Genetic elimination of Cdk5 or blocking ACh production prevents the dispersion of AChR clusters in agrin mutants. Therefore, we propose that ACh negatively regulates neuromuscular synapse formation through a Cdk5-dependent mechanism.

p75 Is Required for the Establishment of Postnatal Sensory Neuron Diversity by Potentiating Ret Signaling.

Producing the neuronal diversity required to adequately discriminate all elements of somatosensation is a complex task during organogenesis. The mechanisms guiding this process during dorsal root ganglion (DRG) sensory neuron specification remain poorly understood. Here, we show that the p75 neurotrophin receptor interacts with Ret and its GFRα co-receptor upon stimulation with glial cell line-derived neurotrophic factor (GDNF). Furthermore, we demonstrate that p75 is required for GDNF-mediated Ret activation, survival, and cell surface localization of Ret in DRG neurons. In mice in which p75 is deleted specifically within sensory neurons beginning at E12.5, we observe that approximately 20% of neurons are lost between P14 and adulthood, and these losses selectively occur within a subpopulation of Ret+ nonpeptidergic nociceptors, with neurons expressing low levels of Ret impacted most heavily. These results suggest that p75 is required for the development of the nonpeptidergic nociceptor lineage by fine-tuning Ret-mediated trophic support.

Defective neuromuscular synapses in mice lacking amyloid precursor protein (APP) and APP-Like protein 2.

Biochemical and genetic studies place the amyloid precursor protein (APP) at the center stage of Alzheimer's disease (AD) pathogenesis. Although mutations in the APP gene lead to dominant inheritance of familial AD, the normal function of APP remains elusive. Here, we report that the APP family of proteins plays an essential role in the development of neuromuscular synapses. Mice deficient in APP and its homolog APP-like protein 2 (APLP2) exhibit aberrant apposition of presynaptic marker proteins with postsynaptic acetylcholine receptors and excessive nerve terminal sprouting. The number of synaptic vesicles at presynaptic terminals is dramatically reduced. These structural abnormalities are accompanied by defective neurotransmitter release and a high incidence of synaptic failure. Our results identify APP/APLP2 as key regulators of structure and function of developing neuromuscular synapses.

Aberrant patterning of neuromuscular synapses in choline acetyltransferase-deficient mice.

In this study we examined the developmental roles of acetylcholine (ACh) by establishing and analyzing mice lacking choline acetyltransferase (ChAT), the biosynthetic enzyme for ACh. As predicted, ChAT-deficient embryos lack both spontaneous and nerve-evoked postsynaptic potentials in muscle and die at birth. In mutant embryos, abnormally increased nerve branching occurs on contact with muscle, and hyperinnervation continues throughout subsequent prenatal development. Postsynaptically, ACh receptor clusters are markedly increased in number and occupy a broader muscle territory in the mutants. Concomitantly, the mutants have significantly more motor neurons than normal. At an ultrastructural level, nerve terminals are smaller in mutant neuromuscular junctions, and they make fewer synaptic contacts to the postsynaptic muscle membrane, although all of the typical synaptic components are present in the mutant. These results indicate that ChAT is uniquely essential for the patterning and formation of mammalian neuromuscular synapses.

Loss of motoneuron-specific microRNA-218 causes systemic neuromuscular failure.

Dysfunction of microRNA (miRNA) metabolism is thought to underlie diseases affecting motoneurons. One miRNA, miR-218, is abundantly and selectively expressed by developing and mature motoneurons. Here we show that mutant mice lacking miR-218 die neonatally and exhibit neuromuscular junction defects, motoneuron hyperexcitability, and progressive motoneuron cell loss, all of which are hallmarks of motoneuron diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. Gene profiling reveals that miR-218 modestly represses a cohort of hundreds of genes that are neuronally enriched but are not specific to a single neuron subpopulation. Thus, the set of messenger RNAs targeted by miR-218, designated TARGET(218), defines a neuronal gene network that is selectively tuned down in motoneurons to prevent neuromuscular failure and neurodegeneration.

Nestin negatively regulates postsynaptic differentiation of the neuromuscular synapse.

Positive and negative regulation of neurotransmitter receptor aggregation on the postsynaptic membrane is a critical event during synapse formation. Acetylcholine (ACh) and agrin are two opposing signals that regulate ACh receptor (AChR) clustering during neuromuscular junction (NMJ) development. ACh induces dispersion of AChR clusters that are not stabilized by agrin via a cyclin-dependent kinase 5 (Cdk5)-mediated mechanism, but regulation of Cdk5 activation is poorly understood. We found that the intermediate filament protein nestin physically interacts with Cdk5 and is required for ACh-induced association of p35, the co-activator of Cdk5, with the muscle membrane. Blockade of nestin-dependent signaling inhibited ACh-induced Cdk5 activation and the dispersion of AChR clusters in cultured myotubes. Similar to the effects of Cdk5 gene inactivation, knockdown of nestin in agrin-deficient mouse embryos substantially restored AChR clusters. These results suggest that nestin is required for ACh-induced, Cdk5-dependent dispersion of AChR clusters during NMJ development.

Resultados de la suspensión del tratamiento en la leucemia linfoide aguda del niño: seguimiento de 52 casos / Results of termination of treatment in acute lymphoid leukemia in the child: follow-up of 52 cases

Se analiza la evolución de 52 pacientes que padecían de leucemia linfoide aguda (LLA) diagnosticados entre 1969 y 1985, la mayoría de los cuales estaban incluidos en distintos protocolos del grupo latinoamericano de Tratamiento de Hemopatías Malignas (GLATHEM). La muestra constituye la totalidad en pacientes en quienes fue posible suspender la terapéutica en ese periodo. De un total de 118 menores de 15 años incluidos en el GLATHEM, suspendieron el tratamiento 49 (44,5%). Con los protocolos más recientes, el porcentaje se elevó al 60%. Los factores de mal pronóstico más importantes al comienzo de la enfermedad, fueron la leucocitosis superior a 50 x 10 9/L. La coloración de PAS positiva en las células blásticas y el sexo masculino. Más del 60% de todos los pacientes de buen pronóstico pudieron concluir la terapéutica y sólo el 12,5 de aquellos con años, se han producido 8 recaídas (15,5%). No se han producido recaídas después de los 4 años de concluida la terapéutica. De los pacientes que recayeron, 5 tuvieron remisiones prolongadas y se les suspendió nuevamente la terapéutica. Sobreviven 50 pacientes, 25 de ellos por más de 10 años

Infiltración ovárica en la leucemia aguda linfoblástica: comunicación en tres pacientes / Ovarian infiltration in acute lymphoblastic leukemia: report of 3 patients

La infiltración leucémica de los ovarios se ha descrito en varios estudios con una incidencia del 20 al 50% en pacientes que fallecen en la recaida de la leucemia aguda linfoblástica (LAL), pero sólo se ha comunicado esporádicamente durante el curso clínico de la enfermedad. En este trabajo se presentan 3 niñas que tivieron una infiltración ovárica aislada sin recaída hematológica. Dos de las pacientes se hallaban sin tratamiento en el momento de la recaída, la cual ocurrió tardíamente en el curso de la enfermedad. Una de la niñas está clínica y hematológicamente bien en 30 meses después. A medida que se prolonga la supervivencia de los enfermos con LAL, es posible que aumente la frecuencia de esta complicación

Variaciones fisiológicas de la hemoglobina en escolares / Physiological variations of the hemoglobin in scholars

Fueron estudiados 58 niños seleccionados aleatoriamente de la matrícula total de la Escuela Primaria "María Dámasa Jova" de la ciudad de Santa Clara. A cada niño se le realizó determinaciones de hemoglobina, hematocrito, constantes corpusculares, hierro sérico, recuento de reticulocitos, capacidad de saturación de la transferrina y lámina periférica. Del total de niños elegidos, se seleccionaron los que reunieron condiciones para considerarlo no enfermo. El valor medio de Hb encontrado fue de 13,37 gr/dl en la muestra total y 14,04 gr/dl en la seleccinada. El hierro sérico tuvo un valor medio de 86,36 u cg/dl en la muestra total y 92,41 en la seleccionada . No se encontró ningún niño con Hb inferior a 11,4 gr/dl y sólo en uno se hizo el diagnóstico de anemia ferripriva. Se establecieron relaciones enter los ÿ antecedentes de enfermedad, familiares y personales con la ÿ hemoglobina y el hierro sérico de los niños

Estudio de los factores predisponentes en la anemia ferritiva en el niño menor de 1 año: labor educativa del personal de enfermería / Study of predisposing factors in iron deficiency anemia in the child under one year old: educational work of the nursing personnel

Se realizó un estudio para demostrar la influencia que tienen determinados factores relacionados con la madre y el niño en la aparición de la anemia ferritiva mediante la aplicación de una encuesta a 50 madres de niños menores de 1 año diagnosticados con esta enfermedad y que son atendidos en el Hospital Provincial Pediátrico Docente "José Luis Miranda" de Villa Clara. Los datos se procesaron por tabulación manual y se presentaron en tablas estadísticas. Entre los antecedentes màs frecuentes se encuentran: la madre que durante el embarazo presentó anemia (50%) y el parto pretérmino (32%). Están presentes también la anemia en el período de recién nacido (26%), así como una inadecuada ablactaciòn (60%). En estos 2 últimos aspectos es donde màs incide la labor de enfermería, encaminada a lograr una correcta lactancia materna y ablactación del niño en el primer año de vida

Algunos aspectos relacionados con la prevalencia del déficit de hierro en la provincia de Villa Clara / Some aspects related to prevalence of iron deficit in Villa Clara Province

Se analizan 4 estudios realizados en la provincia de Villa Clara relacionados con la prevalencia de déficit de hierro en la infancia. Se aprecia que el valor medio de hemoglobina (Hb) en el lactante osciló entre 10,5 y 11,4 g/dL, en tanto que en los niños de 1 a 5 y de 6 a 14 años, éste fue de 12,1 y 12,5 g/dL, respectivamente. Se señala que la deficiencia de hierro disminuyó progresivamente a medida que aumentó la edad. Se indica que la prevalencia de anemia ferripriva, durante los 2 primeros años de la vida, alcanzó proporciones entre el 22,2 y el 45,5 % en los diferentes estudios. Se informa que la relación entre las malas condiciones socioculturales y la anemia es manifiesta. Se encontró relación entre la anemia durante el embarazo y el hierro sérico en los lactantes, no así con respecto a las cifras de Hb de éstos

Determinación de las cifras normales de hemoglobina en una población de 6 a 14 años de la Ciudad de Santa Clara / Determination of normal hemoglobin figures in a population aged 6-14 years in Santa Clara city

Mediante el uso de una técnica de muestreo aleatorio simple no restringido y sin remplazamiento, se escogieron 132 niños de 6 a 14 años de edad del sector 1 del Policlínico "José R. León Acosta" de Santa Clara. A cada niño se le realizaron determinaciones de hemoglobina (Hb), constantes corpusculares, hierro sérico, conteo de reticulocitos, lámina periférica y heces fecales seriadas. De esta muestra se seleccionaron 50 niños sin alteraciones en sangre periférica y sin parasitismo intestinal, y se constituyó una muestra de niños normales a los efectos de este estudio. La distribución de frecuencias de la Hb en ambas muestras fue normal. Se determinó el valor medio de la Hb en ambas muestras, y no se encontraron diferencias significativas en la media de ambos sexos. Se demostró que 12,0 g/dL es el valor mínimo normal promedio esperado para la Hb en este grupo de edad, por debajo del cual es posible la anemia, y 11,35 g/dL es el límite inferior, por debajo del cual la existencia de esta enfermedad es casi obligada. Pudo demostrarse que en la población objeto de estudio la existencia de anemia no constituye un problema