Both GEF domains of the autism and developmental epileptic encephalopathy-associated Trio protein are required for proper tangential migration of GABAergic interneurons.

Recessive and de novo mutations in the TRIO gene are associated with intellectual deficiency (ID), autism spectrum disorder (ASD) and developmental epileptic encephalopathies (DEE). TRIO is a dual guanine nucleotide exchange factor (GEF) that activates Rac1, Cdc42 and RhoA. Trio has been extensively studied in excitatory neurons, and has recently been found to regulate the switch from tangential to radial migration in GABAergic interneurons (INs) through GEFD1-Rac1-dependent SDF1α/CXCR4 signaling. Given the central role of Rho-GTPases during neuronal migration and the implication of IN pathologies in ASD and DEE, we investigated the relative roles of both Trio's GEF domains in regulating the dynamics of INs tangential migration. In Trio-/- mice, we observed reduced numbers of tangentially migrating INs, with intact progenitor proliferation. Further, we noted increased growth cone collapse in developing INs, suggesting altered cytoskeleton dynamics. To bypass the embryonic mortality of Trio-/- mice, we generated Dlx5/6Cre;Trioc/c conditional mutant mice (TriocKO), which develop spontaneous seizures and behavioral deficits reminiscent of ASD and ID. These phenotypes are associated with reduced cortical IN density and functional cortical inhibition. Mechanistically, this reduction of cortical IN numbers reflects a premature switch to radial migration, with an aberrant early entry in the cortical plate, as well as major deficits in cytoskeletal dynamics, including enhanced leading neurite branching and slower nucleokinesis reflecting reduced actin filament condensation and turnover as well as a loss of response to the motogenic effect of EphA4/ephrin A2 reverse signaling. Further, we show that both Trio GEFD1 and GEFD2 domains are required for proper IN migration, with a dominant role of the RhoA-activating GEFD2 domain. Altogether, our data show a critical role of the DEE/ASD-associated Trio gene in the establishment of cortical inhibition and the requirement of both GEF domains in regulating IN migration dynamics.

Exosome secretion is a key pathway for clearance of pathological TDP-43.

Cytoplasmic TDP-43 aggregation is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here we investigated the role of exosomes in the secretion and propagation of TDP-43 aggregates. TDP-43 was detected in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Evidence is presented that protein aggregation and autophagy inhibition are factors that promote exosomal secretion of TDP-43. We also report that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP-43A315T mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosome production would seem inappropriate, as in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43.

Cholinergic neurons intrinsic to the primate external pallidum.

We report the presence of a small population of cholinergic neurons closely intermingled with external pallidal neurons in cynomolgus monkeys (Macaca fascicularis). The majority of these cholinergic pallidal neurons are devoid of Nerve Growth Factor receptor (NGFr), which sets them apart from the population of corticopetal NGFr-rich neurons of the nucleus basalis of Meynert and its ectopic elements that impinge dorsally upon the pallidum via the medullary laminae.

Quantitative and ultrastructural study of serotonin innervation of the globus pallidus in squirrel monkeys.

The present immunohistochemical study was aimed at characterizing the serotonin (5-HT) innervation of the internal (GPi) and external (GPe) pallidal segments in the squirrel monkey (Saimiri sciureus) with an antibody against the 5-HT transporter (SERT). At the light microscopic level, unbiased counts of SERT+ axon varicosities showed that the density of innervation is similar in the GPi (0.57 ± 0.03 × 10(6)  varicosities/mm(3) of tissue) and the GPe (0.60 ± 0.04 × 10(6) ), with the anterior half of both segments being more densely innervated than the posterior half. Dorsoventral and mediolateral decreasing gradients of SERT varicosities occur in both pallidal segments, but are statistically significant only in the GPi. The neuronal density being significantly greater in the GPe (3.41 ± 0.23 × 10(3)  neurons/mm(3) ) than in the GPi (2.90 ± 0.11 × 103), the number of 5-HT axon varicosities per pallidal neuron was found to be superior in the GPi (201 ± 27) than in the GPe (156 ± 26). At the electron microscopic level, SERT+ axon varicosities are comparable in size and vesicular content in GPi and GPe, where they establish mainly asynaptic contacts with unlabeled profiles. Less than 25% of SERT+ varicosities display a synaptic specialization, which is of the symmetrical or asymmetrical type and occurs exclusively on pallidal dendrites. No SERT+ axo-axonic synapses are present, suggesting that 5-HT exerts its well-established modulatory action upon various pallidal afferents mainly through diffuse transmission, whereas its direct control of pallidal neurons results from both volumic and synaptic release of the transmitter.

Ex Utero Electroporation and Organotypic Slice Cultures of Embryonic Mouse Brains for Live-Imaging of Migrating GABAergic Interneurons.

GABAergic interneurons (INs) are critical components of neuronal networks that drive cognition and behavior. INs destined to populate the cortex migrate tangentially from their place of origin in the ventral telencephalon (including from the medial and caudal ganglionic eminences (MGE, CGE)) to the dorsal cortical plate in response to a variety of intrinsic and extrinsic cues. Different methodologies have been developed over the years to genetically manipulate specific pathways and investigate how they regulate the dynamic cytoskeletal changes required for proper IN migration. In utero electroporation has been extensively used to study the effect of gene repression or overexpression in specific IN subtypes while assessing the impact on morphology and final position. However, while this approach is readily used to modify radially migrating pyramidal cells, it is more technically challenging when targeting INs. In utero electroporation generates a low yield given the decreased survival rates of pups when electroporation is conducted before e14.5, as is customary when studying MGE-derived INs. In an alternative approach, MGE explants provide easy access to the MGE and facilitate the imaging of genetically modified INs. However, in these explants, INs migrate into an artificial matrix, devoid of endogenous guidance cues and thalamic inputs. This prompted us to optimize a method where INs can migrate in a more naturalistic environment, while circumventing the technical challenges of in utero approaches. In this paper, we describe the combination of ex utero electroporation of embryonic mouse brains followed by organotypic slice cultures to readily track, image and reconstruct genetically modified INs migrating along their natural paths in response to endogenous cues. This approach allows for both the quantification of the dynamic aspects of IN migration with time-lapse confocal imaging, as well as the detailed analysis of various morphological parameters using neuronal reconstructions on fixed immunolabeled tissue.

Evidence for Sprouting of Dopamine and Serotonin Axons in the Pallidum of Parkinsonian Monkeys.

This light and electron microscopie immunohistochemical quantitative study aimed at determining the state of the dopamine (DA) and serotonin (5-HT) innervations of the internal (GPi) and external (GPe) segments of the pallidum in cynomolgus monkeys (Macaca fascicularis) rendered parkinsonian by systemic injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In contrast to the prominent DA denervation of striatum, the GPi in MPTP monkeys was found to be markedly enriched in DA (TH+) axon varicosities. The posterior sensorimotor region of this major output structure of the basal ganglia was about 8 times more intensely innervated in MPTP monkeys (0.71 ± 0.08 × 106 TH+ axon varicosities/mm3) than in controls (0.09 ± 0.01 × 106). MPTP intoxication also induced a two-fold increase in the density of 5-HT (SERT+) axon varicosities in both GPe and GPi. This augmentation was particularly pronounced anteriorly in the so-called associative and limbic pallidal territories. The total length of the labeled pallidal axons was also significantly increased in MPTP monkeys compared to controls, but the number of DA and 5-HT axon varicosities per axon length unit remained the same in the two groups, indicating that the DA and 5-HT pallidal hyperinnervations seen in MPTP monkeys result from axon sprouting rather than from the appearance of newly formed axon varicosities on non-growing axons. At the ultrastructural level, pallidal TH+ and SERT+ axons were morphologically similar in MPTP and controls, and their synaptic incidence was very low suggesting a volumic mode of transmission. Altogether, our data reveal a significant sprouting of DA and 5-HT pallidal afferents in parkinsonian monkeys, the functional significance of which remains to be determined. We suggest that the marked DA hyperinnervation of the GPi represents a neuroadaptive change designed to normalize pallidal firing patterns associated with the delayed appearance of motor symptoms, whereas the 5-HT hyperinnervation might be involved in the early expression of non-motor symptoms in Parkinson's disease.

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy.

Despite all the technological advances at the light microscopy level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Chemical anatomy of pallidal afferents in primates.

Neurons of the globus pallidus receive massive inputs from the striatum and the subthalamic nucleus, but their activity, as well as those of their striatal and subthalamic inputs, are modulated by brainstem afferents. These include serotonin (5-HT) projections from the dorsal raphe nucleus, cholinergic (ACh) inputs from the pedunculopontine tegmental nucleus, and dopamine (DA) afferents from the substantia nigra pars compacta. This review summarizes our recent findings on the distribution, quantitative and ultrastructural aspects of pallidal 5-HT, ACh and DA innervations. These results have led to the elaboration of a new model of the pallidal neuron based on a precise knowledge of the hierarchy and chemical features of the various synaptic inputs. The dense 5-HT, ACh and DA innervations disclosed in the associative and limbic pallidal territories suggest that these brainstem inputs contribute principally to the planification of motor behaviors and the regulation of attention and mood. Although 5-HT, ACh and DA inputs were found to modulate pallidal neurons and their afferents mainly through asynaptic (volume) transmission, genuine synaptic contacts occur between these chemospecific axon varicosities and pallidal dendrites, revealing that these brainstem projections have a direct access to pallidal neurons, in addition to their indirect input through the striatum and subthalamic nucleus. Altogether, these findings reveal that the brainstem 5-HT, ACh and DA pallidal afferents act in concert with the more robust GABAergic inhibitory striatopallidal and glutamatergic excitatory subthalamopallidal inputs. We hypothesize that a fragile equilibrium between forebrain and brainstem pallidal afferents plays a key role in the functional organization of the primate basal ganglia, in both health and disease.

Asynaptic feature and heterogeneous distribution of the cholinergic innervation of the globus pallidus in primates.

The internal (GPi) and external (GPe) segments of the primate globus pallidus receive a significant cholinergic (ACh) innervation from the brainstem pedunculopontine tegmental nucleus. The present immunohistochemical study describes this innervation in the squirrel monkey (Saimiri sciureus), as visualized with an antibody raised against choline acetyltransferase (ChAT). At the light microscopic level, unbiased stereological quantification of ChAT positive (+) axon varicosities reveals a significantly lower density of innervation in GPi (0.26 ± 0.03 × 10(6)) than in GPe (0.47 ± 0.07 × 10(6) varicosities/mm(3) of tissue), with the anterior half of both segments more densely innervated than the posterior half. Neuronal density of GPi (3.00 ± 0.13 × 10(3) neurons/mm(3)) and GPe (3.62 ± 0.22 × 10(3) neurons/mm(3)) yields a mean ratio of ChAT+ axon varicosities per pallidal neuron of 74 ± 10 in the GPi and 128 ± 28 in the GPe. At the electron microscopic level, the pallidal ChAT+ axon varicosities are significantly smaller than their unlabeled counterparts, but are comparable in size and shape in the two pallidal segments. Only a minority of ChAT+ varicosities displays a synaptic specialization (12 % in the GPi and 17 % in the GPe); these scarce synaptic contacts are mostly of the symmetrical type and occur exclusively on pallidal dendrites. No ChAT+ axo-axonic synaptic contacts are observed, suggesting that ACh exerts its modulatory action on pallidal afferents through diffuse transmission, whereas pallidal neurons may be influenced by both volumic and synaptic delivery of ACh.

Morphological evidence for dopamine interactions with pallidal neurons in primates.

The external (GPe) and internal (GPi) segments of the primate globus pallidus receive dopamine (DA) axonal projections arising mainly from the substantia nigra pars compacta and this innervation is here described based on tyrosine hydroxylase (TH) immunohistochemical observations gathered in the squirrel monkey (Saimiri sciureus). At the light microscopic level, unbiased stereological quantification of TH positive (+) axon varicosities reveals a similar density of innervation in the GPe (0.19 ± 0.02 × 10(6) axon varicosities/mm(3) of tissue) and GPi (0.17 ± 0.01 × 10(6)), but regional variations occur in the anteroposterior and dorsoventral axes in both GPe and GPi and along the mediolateral plane in the GPe. Estimation of the neuronal population in the GPe (3.47 ± 0.15 × 10(3) neurons/mm(3)) and GPi (2.69 ± 0.18 × 10(6)) yields a mean ratio of, respectively, 28 ± 3 and 68 ± 15 TH+ axon varicosities/pallidal neuron. At the electron microscopic level, TH+ axon varicosities in the GPe appear significantly smaller than those in the GPi and very few TH+ axon varicosities are engaged in synaptic contacts in the GPe (17 ± 3%) and the GPi (15 ± 4%) compared to their unlabeled counterparts (77 ± 6 and 50 ± 12%, respectively). Genuine synaptic contacts made by TH+ axon varicosities in the GPe and GPi are of the symmetrical and asymmetrical type. Such synaptic contacts together with the presence of numerous synaptic vesicles in all TH+ axon varicosities observed in the GPe and GPi support the functionality of the DA pallidal innervation. By virtue of its predominantly volumic mode of action, DA appears to exert a key modulatory effect upon pallidal neurons in concert with the more direct GABAergic inhibitory and glutamatergic excitatory actions of the striatum and subthalamic nucleus. We argue that the DA pallidal innervation plays a major role in the functional organization of the primate basal ganglia under both normal and pathological conditions.

Corrigendum: Morphological evidence for dopamine interactions with pallidal neurons in primates.

[This corrects the article on p. 111 in vol. 9, PMID: 26321923.].

A formação do enfermeiro para a Estratégia Saúde da Família / The training of nurses for the Family Health Strategy / La formación de los enfermeros para la Estrategia de Salud de la Familia

Objetivo: refletir sobre a formação do graduando em Enfermagem, com base no referencial da Estratégia Saúde da Família (ESF). Metodologia: trata-se de uma pesquisa bibliográfica realizada nas bases de dados da Biblioteca Virtual em Saúde (BVS), no portal Scientific Electronic Library Online (SciELO) e Literatura Latino-Americana e do Caribe em Ciências da Saúde (LILACS). Englobou artigos científicos publicados entre 2002 à 2009 em português. Para a seleção dos artigos foram utilizados os descritores: sistema único de saúde; saúde da família; educação em enfermagem. A seleção foi feita utilizando um instrumento construído para este fim. Após as análises, que foram realizadas de forma independente por três pesquisadores, selecionou-se 13 publicações que apresentavam relação com a temática. Resultados: a estratégia saúde da família exige um perfil diferenciado dos profissionais, os quais devem mudar o pólo indivíduo/doença/cura para uma assistência holística, promovendo saúde de forma integral. Nesta perspectiva, fazem-se necessárias reformulações nos currículos de graduação em enfermagem a partir de Projetos Pedagógicos centrados no aluno, falar teoria/prática e, adaptando-se ao perfil epidemiológico ao qual está inserido. Apesar das mudanças na formação do graduando, ainda persistem lacunas e desafios a serem superados. Conclusão: enfatiza-se a importância da introdução precoce de conteúdos acerca da ESF, a fim de promover a formação do enfermeiro para atuar nesta perspectiva interdisciplinar no âmbito do SUS, correspondendo às exigências deste novo perfil da saúde.(AU)

Objective: to think about the formation of the nursing senior student having family health strategy as a base. Methodology: this paper describes a bibliographic research done based on Virtual Health Library (BVS - Biblioteca Virtual em Saúde) on the site Scientific Electronic Library on-line (Scielo) and Latin-America and Caribbean Literature in Health Science (Lilacs), along the period of 2002 to 2009 in Portuguese. For the selection of the articles were the following keywords: national health care system, family health, education, nursing. The selection was made using an instrument constructed for this purpose. After the analysis, which were performed independently by three researchers were selected from 13 publications that had relation with the theme. Result: the Family Health Strategy demand a special profile from the professionals who must change the individual /illness/ cure approach to a holistic assistance promoting health in a integrated way. Under these perspectives it´s necessary a reformulation in the nursing graduation curriculum having projects centered in the student working together theory and practice and adapting to the epidemiology profile in which it is inserted. Although there have been changes for the graduating students, there are still some gaps and challenges to be overcome. Conclusion: the importance of an early introduction of ESF subject is emphasize in order to promote the nurse formation making him/her able to work bearing this interdisciplinary perspective in mind at SUS matching with this new demand of health profile.(AU)

Objetivo: reflexionar sobre la formación de los estudiantes de Enfermería, tomándose por base referencial la Estrategia Salud de la Familia (ESF). Metodología: se trata de una investigación bibliográfica realizada en las bases de datos de la Biblioteca Virtual en Salud (BVS), en los portales Scientific Electronic Library Online (SciELO) y Literatura LatinoAmericana y del Caribe en Ciencias de la Salud (LILACS), durante el período de 2002 a 2009,em portugués. Envolvió artículos científicos publicados entre 2002 a 2009 en portugués. Para la selección de los artículos fueron las siguientes palabras: sistema nacional de salud, salud familiar, la educación, la enfermería. La selección se hizo mediante un instrumento construido para este propósito. Después del análisis, que se realizaron de forma independiente por tres investigadores fueron seleccionados de 13 publicaciones que habían relación con el tema. Resultados: la Estrategia Salud de la Familia exige un perfil diferenciado de los profesionales, quienes deben cambiar el foco de individuo/enfermedad/cura para una atención holística, que promueva la salud de forma integral. Bajo este enfoque, se hacen necesarias alteraciones en los currículos de graduación en enfermería a partir de Proyectos Pedagógicos centrados en el alumno, proyectos estos que unan teoría y práctica, y que se adapten al perfil epidemiológico en el que están insertados. A pesar de los cambios ya realizados en la formación de los estudiantes, aún existen lagunas y retos por superarse. Conclusion: se hace hincapié en la importancia de la introducción precoz de contenidos acerca de la ESF en la formación de los enfermeros, con la finalidad de promocionarles una actuación dentro de esta perspectiva interdisciplinaria en el ámbito de SUS, consonante con las exigencias de este nuevo perfil de salud.(AU)