Probes for FOS

Despite the widespread use of general anaesthetics, the mechanisms mediating their effects are still not understood. Although suppressed in most parts of the brain, neuronal activity, as measured by FOS activation, is increased in the hypothalamic supraoptic nucleus (SON) by numerous general anaesthetics, and evidence points to this brain region being involved in the induction of general anaesthesia and natural sleep. Posttranslational modifications of proteins, including changes in phosphorylation, enable fast modulation of protein function which could be underlying the rapid effects of general anaesthesia. In order to identify potential phosphorylation events in the brain mediating general anaesthesia effects, we have explored the phosphoproteome responses in the rat SON, and compared these to cingulate cortex (CC) which displays no FOS activation is response to general anaesthetics.Adult Sprague-Dawley rats were treated with isoflurane for 15 minutes. Proteins from the CC and SON were extracted and processed for Nano-LC Mass Spectrometry (LC-MS/MS). Phosphoproteomic determinations were performed by LC-MS/MS.We found many changes in the phosphoproteomes of both the CC and SON in response to 15 minutes of isoflurane exposure. Pathway analysis indicated that proteins undergoing phosphorylation adaptations are involved in cytoskeleton remodelling and synaptic signalling events. Importantly, changes in protein phosphorylation appeared to be brain region-specific suggesting that differential phosphorylation adaptations might underlie the different neuronal activity responses to general anaesthesia between the CC and SON.In summary, these data suggest that rapid posttranslational modifications in proteins involved in cytoskeleton remodelling and synaptic signalling events might mediate the central mechanisms mediating general anaesthesia.S. Karger AG, Basel.

The synthetic opioid fentanyl is a major contributor to the current opioid addiction crisis. We report that claustral neurons projecting to the frontal cortex limit oral fentanyl self-administration in mice. We found that fentanyl transcriptionally activates frontal-projecting claustrum neurons. These neurons also exhibit a unique suppression of Ca2+ activity upon initiation of bouts of fentanyl consumption. Optogenetic stimulation of frontal-projecting claustral neurons, intervening in this suppression, decreased bouts of fentanyl consumption. In contrast, constitutive inhibition of frontal-projecting claustral neurons in the context of a novel, group-housed self-administration procedure increased fentanyl bout consumption. This same manipulation also sensitized conditioned-place preference for fentanyl and enhanced the representation of fentanyl experience in the frontal cortex. Together, our results indicate that claustrum neurons exert inhibitory control over frontal cortical neurons to restrict oral fentanyl intake. Upregulation of activity in the claustro-frontal projection may be a promising strategy for reducing human opioid addiction.

The excessive release of the antidiuretic hormone vasopressin is implicated in many diseases including cardiovascular disease, diabetes, obesity, and metabolic syndrome. Once thought to be elevated as a consequence of diseases, data now supports a more causative role. We have previously identified CREB3L1 as a transcription factor that co-ordinates vasopressin synthesis and release in the hypothalamus. The objective here was to identify mechanisms orchestrated by CREB3L1 that co-ordinate vasopressin release.We mined Creb3l1 knockdown SON RNA-seq data to identify downstream target genes. We proceeded to investigate the expression of these genes and associated pathways in the supraoptic nucleus of the hypothalamus in response to physiological and pharmacological stimulation. We used viruses to selectively knockdown gene expression in the supraoptic nucleus and assessed physiological and metabolic parameters. We adopted a phosphoproteomics strategy to investigate mechanisms that facilitate hormone release by the pituitary gland.We discovered glucagon like peptide 1 receptor (Glp1r) as a downstream target gene and found increased expression in stimulated vasopressin neurones. Selective knockdown of supraoptic nucleus Glp1rs resulted in decreased food intake and body weight. Treatment with GLP-1R agonist liraglutide decreased vasopressin synthesis and release. Quantitative phosphoproteomics of the pituitary neurointermediate lobe revealed that liraglutide initiates hyperphosphorylation of presynapse active zone proteins that control vasopressin exocytosis.In summary, we show that GLP-1R signalling inhibits the vasopressin system. Our data advises that hydration status may influence the pharmacodynamics of GLP-1R agonists so should be considered in current therapeutic strategies.

Oligodendrocytes are glial cells that support and insulate axons in the central nervous system through the production of myelin. Oligodendrocytes arise throughout embryonic and early postnatal development from oligodendrocyte precursor cells (OPCs), and recent work demonstrated that they are a transcriptional heterogeneous cell population, but the regional and functional implications of this heterogeneity are less clear. Here, we apply in situ sequencing (ISS) to simultaneously probe the expression of 124 marker genes of distinct oligodendrocyte populations, providing comprehensive maps of the corpus callosum, cingulate, motor, and somatosensory cortex in the brain, as well as gray matter (GM) and white matter (WM) regions in the spinal cord, at postnatal (P10), juvenile (P20), and young adult (P60) stages. We systematically compare the abundances of these populations and investigate the neighboring preference of distinct oligodendrocyte populations.We observed that oligodendrocyte lineage progression is more advanced in the juvenile spinal cord compared to the brain, corroborating with previous studies. We found myelination still ongoing in the adult corpus callosum while it was more advanced in the cortex. Interestingly, we also observed a lateral-to-medial gradient of oligodendrocyte lineage progression in the juvenile cortex, which could be linked to arealization, as well as a deep-to-superficial gradient with mature oligodendrocytes preferentially accumulating in the deeper layers of the cortex. The ISS experiments also exposed differences in abundances and population dynamics over time between GM and WM regions in the brain and spinal cord, indicating regional differences within GM and WM, and we found that neighboring preferences of some oligodendroglia populations are altered from the juvenile to the adult CNS.Overall, our ISS experiments reveal spatial heterogeneity of oligodendrocyte lineage progression in the brain and spinal cord and uncover differences in the timing of oligodendrocyte differentiation and myelination, which could be relevant to further investigate functional heterogeneity of oligodendroglia, especially in the context of injury or disease.