Probes for INS

Sensory experience influences the establishment of neural connectivity through molecular mechanisms that remain unclear. Here, we employ single-nucleus RNA sequencing to investigate the contribution of sensory-driven gene expression to synaptic refinement in the dorsal lateral geniculate nucleus of the thalamus, a region of the brain that processes visual information. We find that visual experience induces the expression of the cytokine receptor Fn14 in excitatory thalamocortical neurons. By combining electrophysiological and structural techniques, we show that Fn14 is dispensable for early phases of refinement mediated by spontaneous activity but that Fn14 is essential for refinement during a later, experience-dependent period of development. Refinement deficits in mice lacking Fn14 are associated with functionally weaker and structurally smaller retinogeniculate inputs, indicating that Fn14 mediates both functional and anatomical rearrangements in response to sensory experience. These findings identify Fn14 as a molecular link between sensory-driven gene expression and vision-sensitive refinement in the brain.

Despite generally being a reinforcing drug of abuse, amphetamine (amph) also produces effects such as hypophagia and conditioned taste avoidance (CTA), which may indicate that amph acts as an aversive homeostatic stressor. Stress-responsive prolactin-releasing peptide (PrRP)-positive noradrenergic and glucagon-like peptide-1 (GLP-1)-positive neurons in the caudal nucleus of the solitary tract (cNTS) are modulated by metabolic state, and are prime candidates for mediating amph-induced hypophagia and CTA. The present study used dual immunolabeling and fluorescent in situ hybridization (RNAscope) to examine acute amph-induced activation of cFos expression in phenotypically-identified cNTS neurons in ad lib-fed vs. overnight-fasted male Sprague Dawley rats. We also examined the impact of food deprivation on amph-induced CTA. Compared to control saline treatment, amph activated significantly more cNTS neurons, including PrRP-negative noradrenergic (NA) neurons, GABAergic neurons, and glutamatergic neurons, but not PrRP or GLP-1 neurons. Amph also increased neural activation within a subset of central cNTS projection targets, including the lateral parabrachial nucleus and central amygdala, but not the paraventricular hypothalamus. Food deprivation did not alter amph-induced neural activation or impact the ability of amph to support CTA. These findings indicate that PrRP-negative NA and other cNTS neurons are recruited by acute amph treatment regardless of metabolic state, and may participate in amph-induced hypophagia and CTA.

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.

The basolateral amygdala (BLA) is a site of convergence of negative and positive stimuli and is critical for emotional behaviors and associations. However, the neural substrate for negative and positive behaviors and relationship between negative and positive representations in the basolateral amygdala are unknown. Here we identify two genetically distinct, spatially segregated populations of excitatory neurons in the mouse BLA that participate in valence-specific behaviors and are connected through mutual inhibition. These results identify a genetically defined neural circuit for the antagonistic control of emotional behaviors and memories.