Indicadores de cálcio e de voltagem codificados geneticamente na detecção de potenciais de ação e inputs sinápticos em cultura de neurônios hipocampais

Recently, genetically encoded optical indicators have emerged as noninvasive tools of high spatial and temporal resolution utilized to monitor the activity of individual neurons and specific neuronal populations. The increasing number of new optogenetic indicators, together with the absence of comparisons under identical conditions, has generated difficulty in choosing the most appropriate protein, depending on the experimental design. Therefore, the purpose of our study was to compare three recently developed reporter proteins: the calcium indicators GCaMP3 and R-GECO1, and the voltage indicator VSFP butterfly1.2. These probes were expressed in hippocampal neurons in culture, which were subjected to patchclamp recordings and optical imaging. The three groups (each one expressing a protein) exhibited similar values of membrane potential (in mV, GCaMP3: -56 ±8.0, R-GECO1: -57 ±2.5; VSFP: -60 ±3.9, p = 0.86); however, the group of neurons expressing VSFP showed a lower average of input resistance than the other groups (in Mohms, GCaMP3: 161 ±18.3; GECO1-R: 128 ±15.3; VSFP: 94 ±14.0, p = 0.02). Each neuron was submitted to current injections at different frequencies (10 Hz, 5 Hz, 3 Hz, 1.5 Hz, and 0.7 Hz) and their fluorescence responses were recorded in time. In our study, only 26.7% (4/15) of the neurons expressing VSFP showed detectable fluorescence signal in response to action potentials (APs). The average signal-to-noise ratio (SNR) obtained in response to five spikes (at 10 Hz) was small (1.3 ± 0.21), however the rapid kinetics of the VSFP allowed discrimination of APs as individual peaks, with detection of 53% of the evoked APs. Frequencies below 5 Hz and subthreshold signals were undetectable due to high noise. On the other hand, calcium indicators showed the greatest change in fluorescence following the same protocol (five APs at 10 Hz). Among the GCaMP3 expressing neurons, 80% (8/10) exhibited signal, with an average SNR value of 21 ±6.69 (soma), while for the R-GECO1 neurons, 50% (2/4) of the neurons had signal, with a mean SNR value of 52 ±19.7 (soma). For protocols at 10 Hz, 54% of the evoked APs were detected with GCaMP3 and 85% with R-GECO1. APs were detectable in all the analyzed frequencies and fluorescence signals were detected from subthreshold depolarizations as well. Because GCaMP3 is the most likely to yield fluorescence signal and with high SNR, some experiments were performed only with this probe. We demonstrate that GCaMP3 is effective in detecting synaptic inputs (involving Ca2+ influx), with high spatial and temporal resolution. Differences were also observed between the SNR values resulting from evoked APs, compared to spontaneous APs. In recordings of groups of cells, GCaMP3 showed clear discrimination between activated and silent cells, and reveals itself as a potential tool in studies of neuronal synchronization. Thus, our results indicate that the presently available calcium indicators allow detailed studies on neuronal communication, ranging from individual dendritic spines to the investigation of events of synchrony in neuronal networks genetically defined. In contrast, studies employing VSFPs represent a promising technology for monitoring neural activity and, although still to be improved, they may become more appropriate than calcium indicators, since neurons work on a time scale faster than events of calcium may foresee

Concepções e práticas dos profissionais que atuam na educação infantil diante da violência doméstica contra crianças de zero a cinco anos

El estudio presenta el problema de la violencia doméstica contra los niños de hasta cinco años en el contexto de los centros municipales de Educación Infantil (CMEI) en la ciudad de Natal-RN. Constituye un análisis bajo el enfoque teórico y metodológico de carácter cualitativo, en vista de la totalidad, con la asunción de la violencia doméstica contra los niños en su guía sociales, legales, históricos y culturales con el tema. Tiene como objetivo investigar si los profesionales que trabajan en Educación Infantil son capaces de identificar las posibles situaciones de violencia doméstica contra los niños que están bajo su responsabilidad, e incluso si, en los casos de los casos sospechosos o reales de la identidad de saber que las referencias deben ser adoptadas. La ruta entre el conocimiento y el método establecido implica el análisis conceptual de la infancia, la educación infantil y violencia doméstica contra los niños, además de la realización de grupos focales con los participantes de la encuesta con sus registros en un diario de campo. Perciben a sí mismos para el estudio de las contradicciones en la lucha contra la violencia doméstica contra los niños. Incluso con los conocimientos teóricos sobre el tema, los profesionales no pueden dar referencias adecuadas para proteger a los niños y detener la violencia. Nos encontramos con que las condiciones objetivas del trabajo de los profesionales que trabajan en CMEI, asociados con poco conocimiento sobre el tema, no contribuyen a las referencias. Llegamos a la conclusión de que es necesario la participación de la Secretaría Municipal de Educación, sin olvidar que todas las acciones tienen limitaciones como la violencia doméstica contra los niños también se relaciona con problemas estructurales de la sociabilidad capitalista.