A protocol for concurrent high‐quality immunohistochemical and biochemical analyses in adult mouse central nervous system

Notter, Tina; Panzanelli, Patrizia; Pfister, Sandra L.; Mircsof, Dennis; Fritschy, Jean‐Marc

doi:10.1111/ejn.12447

Cited by 57 publications

(43 citation statements)

References 33 publications

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“…For immunoblotting, hippocampi of control mice were isolated and shock frozen on dry ice. For histological analysis the half brains were post-fixed in 4% PFA in PBS (pH = 6.9; Sigma-Aldrich) for 3–4 h at 4°C, under shaking (Notter et al, 2014). Hemispheres were then briefly washed with PBS and cryoprotected by incubation in 20% sucrose solution for up to 2 days.…”

Section: Methodsmentioning

confidence: 99%

Cannabinoid 1 Receptor Signaling on Hippocampal GABAergic Neurons Influences Microglial Activity

Ativie¹,

Komorowska²,

Beins³

et al. 2018

Front. Mol. Neurosci.

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Microglia, the resident immune cells of the brain, play important roles in defending the brain against pathogens and supporting neuronal circuit plasticity. Chronic or excessive pro-inflammatory responses of microglia damage neurons, therefore their activity is tightly regulated. Pharmacological and genetic studies revealed that cannabinoid type 1 (CB1) receptor activity influences microglial activity, although microglial CB1 receptor expression is very low and activity-dependent. The CB1 receptor is mainly expressed on neurons in the central nervous system (CNS)—with an especially high level on GABAergic interneurons. Here, we determined whether CB1 signaling on this neuronal cell type plays a role in regulating microglial activity. We compared microglia density, morphology and cytokine expression in wild-type (WT) and GABAergic neuron-specific CB1 knockout mice (GABA/CB1−/−) under control conditions (saline-treatment) and after 3 h, 24 h or repeated lipopolysaccharide (LPS)-treatment. Our results revealed that hippocampal microglia from saline-treated GABA/CB1−/− mice resembled those of LPS-treated WT mice: enhanced density and larger cell bodies, while the size and complexity of their processes was reduced. No further reduction in the size or complexity of microglia branching was detected after LPS-treatment in GABA/CB1−/− mice, suggesting that microglia in naïve GABA/CB1−/− mice were already in an activated state. This result was further supported by correlating the level of microglial tumor necrosis factor α (TNFα) with their size. Acute LPS-treatment elicited in both genotypes similar changes in the expression of pro-inflammatory cytokines (TNFα, interleukin-6 (IL-6) and interleukin 1β (IL-1β)). However, TNFα expression was still significantly elevated after repeated LPS-treatment in WT, but not in GABA/CB1−/− mice, indicating a faster development of tolerance to LPS. We also tested the possibility that the altered microglia activity in GABA/CB1−/− mice was due to an altered expression of neuron-glia interaction proteins. Indeed, the level of fractalkine (CX3CL1), a neuronal protein involved in the regulation of microglia, was reduced in hippocampal GABAergic neurons in GABA/CB1−/− mice, suggesting a disturbed neuronal control of microglial activity. Our result suggests that CB1 receptor agonists can modulate microglial activity indirectly, through CB1 receptors on GABAergic neurons. Altogether, we demonstrated that GABAergic neurons, despite their relatively low density in the hippocampus, have a specific role in the regulation of microglial activity and cannabinoid signaling plays an important role in this arrangement.

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Section: Methodsmentioning

confidence: 99%

Cannabinoid 1 Receptor Signaling on Hippocampal GABAergic Neurons Influences Microglial Activity

Ativie¹,

Komorowska²,

Beins³

et al. 2018

Front. Mol. Neurosci.

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“…Dissociated hippocampal neurons were prepared and maintained as previously described 49 . In situ hybridization was performed using the QuantiGene (QG) ViewRNA kit from Panomics as previously described with the following modifications.…”

Section: Methodsmentioning

confidence: 99%

Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects

Mircsof¹,

Langouët²,

Rio³

et al. 2015

Nat Neurosci

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The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

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“…Animals were decapitated, the spinal cord obtained by hydro-extrusion with ice-cold ACSF and the ipsilateral L5 DRGs were excised. The lumbar section and the DRGs were dropped into ice-cold freshly prepared fixative [4% paraformaldehyde dissolved in 0.15 M sodium phosphate buffer, pH 7.4] and post-fixed during 60 min, rinsed with phosphate buffer (PBS), cryoprotected by 72 h in 30% sucrose in PBS, embedded in OCT compound (Tissue-Tek; Sakura Finetek, Torrance, CA), frozen with dry ice and stored at − 80°C [49]. …”

Section: Methodsmentioning

confidence: 99%

The α5 subunit containing GABAA receptors contribute to chronic pain

Bravo‐Hernández

Corleto

Barragán-Iglesias

et al. 2016

Pain

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It has been recently proposed that α5-subunit containing GABAA receptors (α5-GABAA receptors) that mediate tonic inhibition might be involved in pain. The purpose of this study was to investigate the contribution of α5-GABAA receptors in the loss of GABAergic inhibition and in formalin-, Complete Freund’s adjuvant (CFA)- and L5/L6 spinal nerve ligation-induced long-lasting hypersensitivity. Formalin or CFA injection and L5/L6 spinal nerve ligation produced long-lasting allodynia and hyperalgesia. Moreover, formalin injection impaired the rate-dependent depression (RDD) of the Hofmann reflex. Peripheral and intrathecal pre-treatment or post-treatment with the α5-GABAA receptor antagonist, L-655,708 (0.15–15 nmol) prevented and reversed, respectively, these long-lasting behaviors. Formalin injection increased α5-GABAA receptors mRNA expression in the spinal cord and dorsal root ganglia (DRG) mainly at 3 days. α5-GABAA receptors were localized in the dorsal spinal cord and DRG co-labeling with NeuN, CGRP and IB4 suggesting their presence in peptidergic and non-peptidergic neurons. These receptors were found mainly in small- and medium-size neurons. Formalin injection enhanced α5-GABAA receptors fluorescence intensity in spinal cord and DRG at 3 and 6 days. Intrathecal administration of L-655,708 (15 nmol) prevented and reversed formalin-induced impairment of RDD. These results suggest that α5-GABAA receptors play a role in the loss of GABAergic inhibition and contribute to long-lasting secondary allodynia and hyperalgesia.

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A protocol for concurrent high‐quality immunohistochemical and biochemical analyses in adult mouse central nervous system

Cited by 57 publications

References 33 publications

Cannabinoid 1 Receptor Signaling on Hippocampal GABAergic Neurons Influences Microglial Activity

Cannabinoid 1 Receptor Signaling on Hippocampal GABAergic Neurons Influences Microglial Activity

Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects

The α5 subunit containing GABAA receptors contribute to chronic pain

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