A Vector-Based Method to Analyze the Topography of Glial Networks

Abstract

Anisotropy of tracer-coupled networks is a hallmark in many brain regions. In the past, the topography of these networks was analyzed using various approaches, which focused on different aspects, e.g., position, tracer signal, or direction of coupled cells. Here, we developed a vector-based method to analyze the extent and preferential direction of tracer spreading. As a model region, we chose the lateral superior olive—a nucleus that exhibits specialized network topography. In acute slices, sulforhodamine 101-positive astrocytes were patch-clamped and dialyzed with the GJ-permeable tracer neurobiotin, which was subsequently labeled with avidin alexa fluor 488. A predetermined threshold was used to differentiate between tracer-coupled and tracer-uncoupled cells. Tracer extent was calculated from the vector means of tracer-coupled cells in four 90° sectors. We then computed the preferential direction using a rotating coordinate system and post hoc fitting of these results with a sinusoidal function. The new method allows for an objective analysis of tracer spreading that provides information about shape and orientation of GJ networks. We expect this approach to become a vital tool for the analysis of coupling anisotropy in many brain regions.

Altered Gap Junction Network Topography in Mouse Models for Human Hereditary Deafness

“…To investigate the effect of reduced spontaneous neuronal activity on astrocytic GJ coupling in the auditory brainstem, we chose the LSO as a model region. In previous studies we could show that LSO astrocyte networks are predominantly anisotropic and oriented orthogonally to the tonotopic axis [ 1 , 9 ]. LSO astrocytes were a priori identified using sulforhodamine (SR) 101-labeling [ 37 ].…”

“…The semi-automated intensity-based cell detection [ 9 ] showed that networks did not differ significantly in basic properties between WT and KO models, i.e. cell number (WT: 64 ± 15, n = 24; Ca V 1.3 KO: 54 ± 12, n = 14, p = 0.468; otoferlin KO: 65 ± 14, n = 8, p = 0.129; Figure 3 Da), area (WT: 0.043 ± 0.009 mm 2 , n = 24; Ca V 1.3 KO: 0.039 ± 0.007 mm 2 , n = 14, p = 0.513; otoferlin KO: 0.036 ± 0.007 mm 2 , n = 8, p = 0.644; Figure 3 Db), and cell density (WT: 1484 ± 331 cells/mm 2 , n = 24; Ca V 1.3 KO: 1398 ± 331 cells/mm 2 , n = 14, p = 0.493; otoferlin KO: 1816 ± 384 cells/mm 2 , n = 8, p = 0.137; Figure 3 Dc).…”

“…LSO astrocyte networks are predominantly orthogonal to the tonotopic axis [ 1 , 9 ]. We next analyzed, if this preferential orientation is maintained in KO models.…”

“…We next analyzed, if this preferential orientation is maintained in KO models. Network anisotropy was analyzed using our vector-based approach with subsequent meta-analysis [ 9 ]. Therefore, we applied a sinusoidal fit to the data to calculate the shape ( R max ) and orientation ( α ) relative to the dorsoventral axis ( Figure 4 Aa,Ba,Ca).…”

“…In the barrel cortex and the barreloid thalamus, tracer coupling is restricted to the barrels [ 7 , 8 ]. Moreover, anisotropic tracer spread is present in the lateral superior olive (LSO) and the inferior colliculus (IC) [ 1 , 2 , 9 ]—two nuclei of the auditory brainstem, in which tracer-coupled networks are oriented orthogonally to the tonotopic axis. Both LSO and IC are tonotopically organized [ 10 , 11 , 12 ] and principal neurons refer to this organization, as their dendritic trees exhibit a narrow morphology with an orientation orthogonal to the tonotopic axis [ 13 , 14 , 15 , 16 , 17 ].…”