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We thank Merche Rivas, Maria Marsal, Gustavo Castro, Jordi Cortes, Alina Hirschmann, and Angel Sandoval (ICFOInstitut de Ciencies Fotoniques) and Mercedes Alba, Marija Radosevic, David Soto, Xavier Gasull, Mar Guasp, and Lidia Sabater (IDIBAPS, Hospital Clinic, University of Barcelona) for their technical support and for providing of reagents, and Josep Dalmau and Myrna R. Rosenfeld (IDIBAPS, Hospital Clinic, University of Barcelona) for their critical review of the manuscript and mentorship. This study was funded by Instituto de Salud Carlos III (ISCIII) and co-financed by the European Union, FIS (PI20/00280, J.P.), Fundacio CELLEX (P.L-A.); Ministerio de Economia y Competitividad culturing and studying other developmental tissues at similar stages such as the notochord, neural plate and lateral plate mesoderm.

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Hippocampal Neuronal Cultures to Detect and Study New Pathogenic Antibodies Involved in Autoimmune Encephalitis

Publicat a:Jove-Journal Of Visualized Experiments. (184): e63829- - 2022-06-01 (184), DOI: 10.3791/63829

Autors: Cunquero, Marina; Aguilar, Esther; Loza-Alvarez, Pablo; Planaguma, Jesus

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Over the last 15 years, a new category of antibody-mediated diseases of the central nervous system (CNS) has been characterized and is now defined as "autoimmune encephalitis" (AE). There are currently 17 known AE syndromes, and all are associated with antibodies against the neuronal cell surface or synaptic proteins. The clinical syndromes are complex and vary according to the type of associated antibody. The best-known of these diseases is anti-N-methyl D-aspartate receptor (NMDAR) encephalitis, which is a prominent neuropsychiatric disorder associated with severe memory and behavioral impairments. The associated antibodies react with the GluN1 subunit of the NMDAR at the N-terminal domain. The approach most frequently used for the discovery and characterization of AE antibodies includes the culture of dissociated, fetal, rodent hippocampal neurons. During the process of antibody characterization, live neurons in culture are exposed to patients' serum or CSF, and the detection of reactivity indicates that the serum or CSF samples of the patient contain antibodies against neuronal surface antigens. Hippocampal cultures can also be used to determine whether the antibodies in patients are potentially pathogenic by examining if they cause structural or functional alterations of the neurons. The level of success of these studies depends on the quality of the cultures and on the protocols used to obtain and detect the reactivity of patient samples. This article provides an optimized protocol for primary cell culture of fetal rat hippocampal neurons combined with immunostaining to determine the presence of antibodies in the serum or CSF of patients. An example of how to examine the potential pathogenic effects of NMDAR antibodies using cultured neurons and calcium imaging is also presented.

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