![]() However, on multiple opportunities, leakage of LY into the surrounding medium prevented imaging of adult PhrMNs. Intrapleural injection with Alexa 488-CTB provides highly specific labeling of PhrMNs and allows the focused impalement of numerous cell bodies with a fluorescent-dye containing microelectrode. It is likely that these differences reflect the better dendritic filling with LY and incomplete filling (mainly via intracellular organelles) with CTB. Our average primary dendrite diameter was 5.2 µm, which is larger than that previously reported with CTB-based retrograde labeling techniques (e.g., 2.7 µm). In order to empirically validate the model, however, a greater sample of PhrMNs should be analyzed across different developmental time points. Including dendrites with minimal branching did not change the relationship between primary dendrite diameter and overall dendritic surface area (r 2 = 0.545 for a quadratic model p=0.0002), with a narrow 95% confidence interval for primary dendrite diameters between 2 and 8 µm. It is possible that our criteria for inclusion will overestimate branching and thus dendritic surface area. However, some dendrites showed minimal branching despite considerable length. Thus, only dendritic arbors known to be fully reconstructed were used in the model. In the present study, dendrites were sampled based on their branching into at least tertiary order. In our study, confocal microscopy allowed for accurate reconstruction of dendritic trees along all axes, and therefore direct measurements of PhrMN surface areas were possible without assumptions of dendrite shape (i.e., cylindrical) or taper rate. showed that stereological methods can overestimate PhrMN soma volumes by up to 300% in cases where the relationship between axial and lateral dimensions deviate from shape assumptions (prolate spheroid). However, shape-based assumptions may yield inaccurate results. , dendrites were measured as successions of cylindrical segments, each having a constant diameter and a measured length, and branching points (regions where individual branches are linked to a stem branch) were assumed as being dichotomous. In the surface area estimation model introduced by Burke et al. A fitted quadratic curve ( 5) is shown in Fig. Mean primary dendrite diameters and dendritic arbor surface area were 5.2 ± 1.3 µm and 6,391 ± 1,895 µm2, respectively.Īll three fitting models (linear, quadratic and power) demonstrated significant correlation between dendritic tree surface area (A) and primary dendrite diameter (d 0). 1 shows fuzzygradient volume renderings of dendritic tree objects created from optical sections of adult rat PhrMNs.ĭendritic branching ranged from 3 rd to 7 th order. A quadratic model for estimating dendritic tree surface area based on measurements of primary dendrite diameter was derived (r 2 = 0.932 p 2 nd order) of at least one primary dendritic tree.Įleven full dentritic arbors showing minor mechanical damage were semi-automatically segmented and assigned to objects based on voxel intensities. Following pre-processing, segmentation was semi-automatically performed in 3D, and direct measurements of dendritic surface area were obtained. Confocal microscopy allowed accurate 3D reconstruction of dendritic arbors from adult rat PhrMNs. In this study, a novel method that improves dendritic filling of PhrMNs in lightly-fixed samples was used. However, limitations intrinsic to shape-based estimations and incomplete labeling of dendritic trees by retrograde techniques have hindered systematic approaches to examine dendritic morphology of PhrMNs. Given that 97% of a motoneuron's receptive area is provided by dendrites, dendritic branching and overall extension are physiologically important in determining the output of their synaptic receptive fields. Stereological techniques that rely on morphological assumptions and direct three-dimensional (3D) confocal measurements have been previously used to estimate the dendritic surface areas of phrenic motoneurons (PhrMNs). Escuela de Ingeniería de Antioquia-Universidad CES, Colombia. Mantilla 1,2ġ Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, United States.Ģ Department of Anesthesiology, Mayo Clinic, Rochester, United States.ģ Programa de Ingeniería Biomédica. MODELO DE ARBORIZACIÓN DENDRÍTICA BASADO EN RECONSTRUCCIONES DE MOTONEURONAS FRÉNICAS EN RATAS ADULTAS MODELING DENDRITIC ARBORIZATION BASED ON 3D-RECONSTRUCTIONS OF ADULT RAT PHRENIC MOTONEURONS
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