Probes for INS

Abstract

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, non-cholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed in situ hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, Nmb mRNA. Most (∼75%) RTN neurons express low-to-moderate levels of Nmb and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, Kcnk5 and Gpr4 These Nmb-low RTN neurons also express varying levels of transcripts for Gal, Penk and Adcyap1,and receptors for substance P, orexin, serotonin and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of Nmb mRNA. These Nmb-high RTN neurons do not express Fos after hypercapnia, have low-to-undetectable levels of Kcnk5 or Gpr4 transcripts; they also express Adcyap1, but are essentially devoid of Penk and Gal transcripts. In male rats, Nmb is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, Nmb is a selective marker of the RTN in rodents; Nmb-low neurons, the vast majority, are central respiratory chemoreceptors whereas Nmb-high neurons likely have other functions.SIGNIFICANCE STATEMENTCentral respiratory chemoreceptors regulate arterial PCO2 by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed in situ hybridization, we show here that a single marker, Neuromedin B mRNA (Nmb), identifies RTN neurons in rodents. We also suggest that >75% of these Nmb neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors (Kcnk5 and Gpr4). The other RTN neurons express very high levels of Nmb, but low levels of Kcnk5/Gpr4/pre-pro-galanin/pre-pro-enkephalin, and do not respond to hypercapnia. Their function is unknown.

A wealth of data has elucidated the mechanisms by which sensory inputs are encoded in the neocortex, but how these processes are regulated by the behavioral relevance of sensory information is less understood. Here, we focus on neocortical layer 1 (L1), a key location for processing of such top-down information. Using Neuron-Derived Neurotrophic Factor(NDNF) as a selective marker of L1 interneurons (INs) and in vivo 2-photon calcium imaging, electrophysiology, viral tracing, optogenetics, and associative memory, we find that L1 NDNF-INs mediate a prolonged form of inhibition in distal pyramidal neuron dendrites that correlates with the strength of the memory trace. Conversely, inhibition from Martinotti cells remains unchanged after conditioning but in turn tightly controls sensory responses in NDNF-INs. These results define a genetically addressable form of dendritic inhibition that is highly experience dependent and indicate that in addition to disinhibition, salient stimuli are encoded at elevated levels of distal dendritic inhibition.

The red nucleus (RN) is required for limb control, specifically fine motor coordination. There is some evidence for a role of the RN in reaching and grasping, mainly from lesion studies, but results so far have been inconsistent. In addition, the role of RN neurons in such learned motor functions at the level of synaptic transmission has been largely neglected. Here, we show that Vglut2-expressing RN neurons undergo plastic events and encode the optimization of fine movements. RN light-ablation severely impairs reaching and grasping functions while sparing general locomotion. We identify a neuronal population co-expressing Vglut2, PV and C1QL2, which specifically undergoes training-dependent plasticity. Selective chemo-genetic inhibition of these neurons perturbs reaching and grasping skills. Our study highlights the role of the Vglut2-positive rubral population in complex fine motor tasks, with its related plasticity representing an important starting point for the investigation of mechanistic substrates of fine motor coordination training.