Characterization of panglial gap junction networks in the thalamus and hippocampus reveals glial heterogeneity

Abstract

Increasing evidence over the past decades revealed the importance of glia in the brain. In this study, astrocytes in the ventrobasal thalamus were characterized in detail for the first time. Glial heterogeneity was investigated by comparing their properties with those of astrocytes in the hippocampus, a brain region in which glial cells have been widely studied.<br /> During development, astrocytes establish gap junction channels with each other. In this study, the networks in the ventrobasal thalamus increased in size until the end of the second postnatal week. Cx30 expression was strongly upregulated during postnatal development and had a predominating role in glial network formation in the mature thalamus. Cx30 and Cx43, but not Cx26, mediated gap junction coupling in the hippocampus and thalamus. Strikingly, some astrocytes were devoid of Cx43. Expression of Cx43 in RG-like cells influences proliferation. The effect of two Cx43 mutations on network sizes were studied to identify the key function, gap junction coupling or adhesion, for proliferation. The function remained elusive. In summary, connexin expression differs among brain regions revealing glial heterogeneity throughout the brain.<br /> Tracer diffusion from astrocytes into the myelin sheath was observed in the ventrobasal thalamus. Such functional gap junction channels between astrocytes and oligodendrocytes were so far unknown. In this study, a panglial network formed by oligodendrocytes and astrocytes was discovered in the hippocampus and the thalamus. Employing Cx30/47 DKO mice, a major impact of Cx30 on panglial network formation was observed in the thalamus. Cx30 and Cx47 do not form functional channels<i>in vitro</i>. This leads to the conclusion that panglial coupling is mainly mediated by Cx30:Cx32 channels.<br /> Immunohistochemical analysis in PLP-GFP and Cx43-ECFP mice identified overlapping protein expression of "classical" markers in thalamic astrocytes and oligodendrocytes in contrast to hippocampal glia. S100β was the most reliable marker to distinguish astrocytes and oligodendrocytes in the ventrobasal thalamus. Unexpectedly, an "intermediate" cell-type was identified co-expressing Cx43 and Olig2. It is a mature cell-type which is part of thalamic glial networks and has been described for the first time in this study.<br /> The functional role of panglial networks in metabolite supply to neurons was analysed in the present study. Glucose diffusion from astrocytes to oligodendrocytes was demonstrated in the thalamus employing the fluorescent glucose analogue 2-NBDG in PLP-GFP mice with SR101 labelled astrocytes. For further investigation of glia-neuron interactions, a method to analyse neuronal field potentials in the ventrobasal thalamus was established and characterized. Extracellular glucose deprivation abolished neuronal postsynaptic field potentials, thereby confirming the neuronal requirement of glial energy supply.<br /> In addition to connexin mediated gap junction channels, the expression of ionotropic AMPA and GABA_A receptors was studied in thalamic astrocytes. In the juvenile thalamus, two astrocyte populations were distinguished by the presence or absence of AMPA receptor expression. The GluA2 subunit was abundantly expressed when AMPA receptors were expressed. All astrocytes expressed GABA_A receptors. They were devoid of the α3 subunit and rarely expressed the γ2 subunit required for synaptic GABA_A receptor localization. Instead, all cells expressed the γ1 subunit. These data indicate that glial heterogeneity occurs even within a given brain regions.<br /> The present study describes panglial networks in grey matter and their distinct properties among brain regions for the first time. In addition, glial heterogeneity was observed between and within brain regions and enhanced our understanding of glial specializations. In addition, a new "intermediate" cell-type was discovered which is abundantly present in the ventrobasal thalamus.