Neuron-astrocyte metabolic coupling facilitates spinal plasticity and maintenance of inflammatory pain.

Long-lasting pain stimuli can trigger maladaptive changes in the spinal cord, reminiscent of plasticity associated with memory formation. Metabolic coupling between astrocytes and neurons has been implicated in neuronal plasticity and memory formation in the central nervous system, but neither its involvement in pathological pain nor in spinal plasticity has been tested. Here we report a form of neuroglia signalling involving spinal astrocytic glycogen dynamics triggered by persistent noxious stimulation via upregulation of the Protein Targeting to Glycogen (PTG) in spinal astrocytes. PTG drove glycogen build-up in astrocytes, and blunting glycogen accumulation and turnover by Ptg gene deletion reduced pain-related behaviours and promoted faster recovery by shortening pain maintenance in mice. Furthermore, mechanistic analyses revealed that glycogen dynamics is a critically required process for maintenance of pain by facilitating neuronal plasticity in spinal lamina 1 neurons. In summary, our study describes a previously unappreciated mechanism of astrocyte-neuron metabolic communication through glycogen breakdown in the spinal cord that fuels spinal neuron hyperexcitability.

Characterization of Posttranslational Modifications on Histone Variants.

The study of histone variants and histone posttranslational modifications (PTMs) is a trending topic in different fields including developmental biology, neurobiology, and immunology; as well as in the understanding of molecular mechanisms leading to diverse diseases, such as cancer. Since the establishment of histone PTMs starts immediately after their synthesis and it continues once they are assembled into chromatin, here we describe a classic protocol of subcellular fractionation aiming to study histones at different stages of maturation. This includes newly synthesized histones enriched in cytosolic fractions; a pool of newly synthesized, evicted, and stored histones enriched in nuclear soluble fractions; and chromatin-associated histones enriched in chromatin pellet. To study specific histone variants and the establishment of their PTMs, we describe a protocol for obtaining histone variants expressed in bacteria. In addition, we describe a Triton-Acetic acid-Urea (TAU) gel electrophoresis protocol adapted to work on mini-gels, which can be coupled to Western blot to analyze PTMs on histone variants. Finally, we describe a Chromatin immunoprecipitation (ChIP) assay for studying histone PTMs, or tagged histone variants, on specific DNA sequences.