Abstract
The activity of excitatory neurons is controlled by a highly diverse population of inhibitory interneurons. These cells show a high level of physiological, morphological and neurochemical heterogeneity, and play highly specific roles in neuronal circuits. In the mammalian hippocampus, these are divided into 21 different subtypes of GABAergic interneurons based on their expression of different markers, morphology and their electrophysiological properties. Ideally, all can be marked using an antibody directed against the inhibitory neurotransmitter GABA, but parvalbumin, calbindin, somatostatin, and calretinin are also commonly used as markers to narrow down the specific interneuron subtype. Here, we describe a journey to find the necessary immunological reagents for studying GABAergic interneurons of the mouse hippocampus. Based on web searches there are several hundreds of different antibodies on the market directed against these four markers. Searches in the literature databases allowed us to narrow it down to a subset of antibodies most commonly used in publications. However, in our hands the most cited ones did not work for immunofluorescence stainings of formaldehyde fixed tissue sections and cultured hippocampal neurons, and we had to immunostain our way through thirteen different commercial antibodies before finally finding a suitable antibody for each of the four markers. The antibodies were evaluated based on signal-to-noise ratios as well as if positive cells were found in layers of the hippocampus where they have previously been described. Additionally, the antibodies were also tested on sections from mouse spinal cord with similar criteria for specificity of the antibodies. Using the antibodies with a high rating on pAbmAbs, an antibody review database, stainings with high signal-to-noise ratios and location of the immunostained cells in accordance with the literature could be obtained, making these antibodies suitable choices for studying the GABAergic system.
Highlights
Hippocampal networks are composed of a large portion of excitatory principal cells and a smaller cohort of inhibitory interneurons[1]
Immunostaining against GABA have shown discrepancy when compared to in-situ hybridization against glutamate decarboxylase, the enzyme that catalyzes the decarboxylation of glutamate to GABA, indicating that some cells may express very low levels of GABA leaving this as an insufficient choice for immunostaining[7,8,9]
Interneurons of the hippocampus Initially, we screened the antibody specificity by staining of cultured hippocampal neurons, evaluating antibodies based on their ability to mark a distinct subset of neurons
Summary
Hippocampal networks are composed of a large portion of excitatory principal cells and a smaller cohort of inhibitory interneurons[1]. Its principal action is mediated through ubiquitous fast ionotropic GABAA receptors by increasing the membrane permeability to Cl- ions[2]. This inhibitory mechanism regulates the excitability of both principal cells and GABAergic interneurons. Immunostaining against GABA have shown discrepancy when compared to in-situ hybridization against glutamate decarboxylase, the enzyme that catalyzes the decarboxylation of glutamate to GABA, indicating that some cells may express very low levels of GABA leaving this as an insufficient choice for immunostaining[7,8,9] These 21 subtypes can be distinguished based on axonal distribution, synaptic targets, neuropeptide or calciumbinding protein content and physiological characteristics[10]. When immunostaining against neuropeptides or calciumbinding proteins, this is not possible, and immunostaining only allows characterization of subgroups
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