Project description:Hunger is a fundamental drive, evolutionarily hard-wired to ensure that an animal has sufficient energy to survive and reproduce. Just as important as knowing when to eat is for an animal to know when not to eat. Here, using spatially resolved, single cell translational phenotyping and ensemble-level molecular profiling, we characterize a small population of neuropeptidergic neurons in the brainstem’s dorsal raphe nucleus (DRN) and describe how they regulate appetite. Together, this work identifies a likely conserved cellular mechanism that transforms diverse neurohumoral signals into a key behavioral output.
Project description:The lateral superior olive (LSO), a conspicuous integration center in the auditory brainstem, contains a remarkably heterogeneous neuron population. Ascending neurons, predominantly principal neurons (pLSOs), process interaural level differences for sound localization. Descending neurons (lateral olivocochlear neurons, LOCs) provide feedback into the cochlea and likely protect from acoustic overexposure. The molecular determinants of the neuronal diversity in the LSO are largely unknown. Here, we employed patch-seq analysis in juvenile mice to classify LSO neurons by their functional and molecular profiles, including developmental aspects. Across the sample (n=86), genes involved in ATPsynthesis were particularly highly expressed, confirming the energy expenditure of auditory neurons. Two clusters were identified, pLSOs and LOCs. They were distinguishable by 353 differentially expressed genes (DEGs), most being novel for the LSO. Electrophysiological analysis corroborated the transcriptomic clustering. We focused on genes impacting neuronal input-output properties and validated some by immunohistochemistry, electrophysiology, and pharmacology. These genes encode proteins like osteopontin, Kv11.3, and Kvb3 (pLSO-specific), calcitonin-gene-related peptide (LOC-specific), or Kv7.2 and Kv7.3 (no DEGs). We identified 12 ‘Super DEGs’ and 12 genes demonstrating ‘Cluster similarity’. Collectively, we provide fundamental and comprehensive insights into the molecular composition of individual ascending and descending neurons in the juvenile auditory brainstem and how this may relate to their specific functions, including developmental aspects.
Project description:To elucidate the molecular mechanism mediating the inactivated effect of DMV neurons on fat absorption, we performed an activity-based protein profiling strategy, using Puerarin as a “bait”. The Puerarin-tag probe was synthesized with a photoreactive tag to enrich and visualize target proteins via a photoaffinity chemistry reaction. We verified that probe-tagged Puerarin retains the same effects of increasing fecal lipid excretion as non-tagged Puerarin. Probe-tagged Puerarin was added to the freshly isolated brainstem sample, and 10 doses of non-tagged Puerarin was used as a competitor of probe-tagged Puerarin. Following the photoaffinity reaction, targeted proteins were subsequently assessed by liquid chromatography tandem mass spectrometry (LC-MS).
Project description:Exaggerated airway constriction triggered by exposure to irritants such as allergen, also called hyperreactivity, is a hallmark of asthma and can be life-threatening. Aside from immune cells, vagal sensory neurons are important for airway hyperreactivity 1-4. However, the identity and signature of the downstream nodes of this adaptive circuit remains poorly understood. Here we show that Dbh+ neurons in the nucleus of the solitary tract (nTS) of the brainstem, and downstream neurons in the nucleus ambiguus (NA), are both necessary and sufficient for chronic allergen-induced airway hyperreactivity. We found that repeated exposures of mice to inhaled allergen activates nTS neurons in a mast cell-, interleukin 4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA-seq followed by RNAscope quantification of the nTS at baseline and following allergen challenges reveals that a Dbh+ population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted, while chemogenetic activation promoted hyperreactivity. Viral tracing indicates that Dbh+ nTS neurons, capable of producing norepinephrine, project to the NA, and NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that then directly drive airway constriction. Focusing on transmitters, delivery of norepinephrine antagonists to the NA blunted allergen-induced hyperreactivity. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. The knowledge opens the possibility of targeting neural modulation as an approach to control refractory allergen-induced airway constriction.
Project description:Exaggerated airway constriction triggered by exposure to irritants such as allergen, also called hyperreactivity, is a hallmark of asthma and can be life-threatening. Aside from immune cells, vagal sensory neurons are important for airway hyperreactivity 1-4. However, the identity and signature of the downstream nodes of this adaptive circuit remains poorly understood. Here we show that Dbh+ neurons in the nucleus of the solitary tract (nTS) of the brainstem, and downstream neurons in the nucleus ambiguus (NA), are both necessary and sufficient for chronic allergen-induced airway hyperreactivity. We found that repeated exposures of mice to inhaled allergen activates nTS neurons in a mast cell-, interleukin 4 (IL-4)- and vagal nerve-dependent manner. Single-nucleus RNA-seq followed by RNAscope quantification of the nTS at baseline and following allergen challenges reveals that a Dbh+ population is preferentially activated. Ablation or chemogenetic inactivation of Dbh+ nTS neurons blunted, while chemogenetic activation promoted hyperreactivity. Viral tracing indicates that Dbh+ nTS neurons, capable of producing norepinephrine, project to the NA, and NA neurons are necessary and sufficient to relay allergen signals to postganglionic neurons that then directly drive airway constriction. Focusing on transmitters, delivery of norepinephrine antagonists to the NA blunted allergen-induced hyperreactivity. Together, these findings provide molecular, anatomical and functional definitions of key nodes of a canonical allergen response circuit. The knowledge opens the possibility of targeting neural modulation as an approach to control allergen-induced airway constriction.
Project description:Leigh Syndrome is characterized by symmetrical brain lesions and neuroinflammation in select nuclei, predominantly brainstem and/or basal ganglia. Accordingly, Ndufs4-deficient mice present overt lesions in brainstem, olfactory bulb, and basal ganglia. To define the contribution of Ndufs4 deficiency in excitatory neurons to the overall neuroinflammatory phenotype, we performed Gene expression analysis in the brainstem of mice with specific deletion of Ndufs4 in Vglut2-expressing neurons (Vglut2:Ndufs4cKO mice). This analysis allowed us to further define the inflammatory phenotype present in the brainstem of Vglut2:Ndufs4cKO mice.