primary-cell-culture-purified-gabaergic-or-glutamatergic-neurons

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...

Universitätsmedizin Berlin

In the mammalian cortex, GABAergic and glutamatergic neurons represent 2 major neuronal classes, which establish inhibitory and excitatory synapses, respectively. Despite differences in their anatomy, physiology and developmental origin, both cell types require support from glial cells, particularly astrocytes, for their growth and survival. Recent experiments indicate that glutamatergic neurons also depend on astrocytes for synapse formation. However, it is not clear if the same holds true for GABAergic neurons. By studying highly pure GABAergic cell cultures, established through fluorescent activated cell sorting, we find that purified GABAergic neurons are smaller and have reduced survival, nevertheless they establish robust synaptic transmission in the absence of glia. Support from glial cells reverses morphological and survival deficits, but does little to alter synaptic transmission. In contrast, in cultures of purified glutamatergic neurons, morphological development, survival and synaptic transmission are collectively dependent on glial support. Thus, our results demonstrate a fundamental difference in the way GABAergic and glutamatergic neurons depend on glia for the establishment of synaptic transmission, a finding that has important implications for our understanding of how neuronal networks develop.

Differential Dependence of GABAergic and Glutamatergic Neurons on Glia for the Establishment of Synaptic Transmission.

The y-aminobutyric acid A receptor (GABAR) participates in most neurophysiological processes. Mutations cause epilepsy and neuropsychiatric pathologies. Recently a severe encephalitis with refractory seizures and antibodies against GABARs has been described. Considering the complex subunit distribution of GABARs, binding patterns of human GABAR antibodies will help to understand the pathophysiology underlying diverse clinical pictures. We therefore investigated the cerebrospinal fluid (CSF) reactivity of a patient with GABAR encephalitis using immunocytochemistry on murine brain sections and compared its specificity with commercial GABAR antibodies. The immunoreactivity of the patient's CSF showed excellent agreement with previously reported GABAR mRNA expression. Colocalization with neuronal and glial markers verified the neuronal specificity of GABAR. Patient antibodies strongly bound to neuropil in the external layers of the olfactory bulb, CA1 and CA2 of the hippocampus, neocortex, pallidum and granular cells of the cerebellum. Distribution patterns suggest the presence of polyclonal CSF GABAR antibodies targeting multiple receptor subunits. The comparison with commercial antibodies revealed large overlap, but also specific differences. For example, commercial antibodies accumulated on dendrites, while CSF created a homogeneous neuropil signal. The number of GABAergic synapses stained with CSF exceeded those labeled with the commercial antibodies. In some areas, commercial antibodies and CSF even stained complementary populations of GABAergic neurons. The data indicate the presence of additional anti-neuronal autoantibodies in the CSF, which could be assessed in future studies with individual recombinant monoclonal antibodies from CSF B cells. This strategy would confirm antibody pathogenicity and likely explain variable clinical pictures in autoimmune encephalitis patients.

Paul Turko

Institut für Integrative Neuroanatomie,
Charité,
Institut für Integrative Neuroanatomie, Charité

Universitätsmedizin Berlin

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

Paul Turko

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Universitätsmedizin Berlin

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

Institut für Integrative Neuroanatomie,
Charité,
Institut für Integrative Neuroanatomie, Charité