Probes for INS

Cocaine and Amphetamine-regulated Transcript (CART) is one of the most abundant neuropeptides in vagal afferents, including those involved in regulating feeding. Recent observations indicate that metabolic challenges dramatically alter the neuropeptidergic profile of CART-producing vagal afferents. Here, using confocal microscopy, we re-assessed the distribution and regulation of CART (55-102) immunoreactivity in vagal afferents of the male mouse in response to metabolic challenges, including fasting, high-fat diet feeding. Importantly, the perikarya and axons of vagal C-fibers were labeled using mice expressing channelrodhopsin-2 (ChR2-YFP) in Nav1.8-Cre-expressing neurons. In these mice, approximately 82% of the nodose ganglion neurons were labeled with ChR2-YFP. Furthermore, ChR2-YFP-labeled axons could easily be identified in the dorsovagal complex. CART (55-102) immunoreactivity was observed in 55% of the ChR2-YFP-labeled neurons in the nodose ganglion and 22% of the ChR2-YFP-labeled varicosities within the area postrema of fed, fasted and obese mice. The distribution of positive profiles was also identical across the full range of CART staining in fed, fasted and obese mice. In contrast to previous studies, fasting did not induce melanin-concentrating hormone immunoreactivity in vagal afferents. Moreover, prepro-MCH mRNA was undetectable in the nodose ganglion of fasted mice. In summary, this study showed that the perikarya and central terminals of vagal afferents are invariably enriched in CART and devoid of MCH.

Significance Statement Recent studies reported that fasting triggers vagal afferents to switch from expressing anorectic to orexigenic neuropeptides. This study failed to replicate the aforementioned observations using a combination of confocal microscopy, immunohistochemistry, and in situ hybridization. In particular, we showed that neither fasting nor diet-induced obesity influence the immunoreactivity for Cocaine and Amphetamine-regulated Transcript neuropeptide in the mouse vagal afferents. In contrast to previous studies, we also failed to detect melanin-concentrating hormone expression in the mouse vagal afferents. Overall, we reached the conclusion that the neuropeptidergic profile of the vagal afferents involved in feeding is remarkably stable in response to metabolic challenges.

The goals of this study were to localize the neuropeptide Cocaine- and Amphetamine-Regulated Transcript (CART) in primate retinas and to describe the morphology, neurotransmitter content and synaptic connections of the neurons that contain it. Using in situ hybridization, light and electron microscopic immunolabeling, CART was localized to GABAergic amacrine cells in baboon retinas. The CART-positive cells had thin, varicose dendrites that gradually descended through the inner plexiform layer and ramified extensively in the innermost stratum. They resembled two types of wide-field diffuse amacrine cells that had been described previously in macaque retinas using the Golgi method and also A17, serotonin-accumulating and waterfall cells of other mammals. The CART-positive cells received synapses from rod bipolar cell axons and made synapses onto the axons in a reciprocal configuration. The CART-positive cells also received synapses from other amacrine cells. Some of these were located on their primary dendrites, and the presynaptic cells there included dopaminergic amacrine cells. Although some CART-positive somas were localized in the ganglion cell layer, they did not contain the ganglion cell marker RNA binding protein with multiple splicing (RBPMS). Based on these results and electrophysiological studies in other mammals, the CART-positive amacrine cells would be expected to play a major role in the primary rod pathway of primates, providing feedback inhibition to rod bipolar cells.

The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. Two neuronal cell types found in the LHA are defined by their expression of hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH) and are both important regulators of arousal, feeding and metabolism. Conflicting evidence suggests that these cell populations have a more complex signaling repertoire than previously appreciated, particularly in regard to their co-expression of other neuropeptides and the machinery for the synthesis and release of GABA and glutamate. Here, we undertook a single cell expression profiling approach to decipher the neurochemical phenotype, and heterogeneity therein, of Hcrt/Ox and MCH neurons. In transgenic mouse lines, we used single cell qPCR to quantify the expression of 48 key genes, which include neuropeptides, fast neurotransmitter components and other key markers, which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of a vesicular GABA release pathway. Furthermore, we found that this profile is characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs.

Significance Statement The lateral hypothalamic area (LHA) is a key regulator of fundamental behavioral states such as arousal, stress and reward, and disruption of neural circuits in this region is associated with disorders of sleep, feeding and motivated behavior. The multifunctional nature of the LHA is attributable to a heterogeneous population of neurons that exhibit significant phenotypic and neurochemical diversity. Here we sought to resolve aspects of this diversity in two well-studied but incompletely understood LHA neuron populations, defined by their expression of neuropeptides hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH). These efforts lay a foundation for understanding, at a molecular and cellular level, how Hcrt/Ox and MCH neurons coordinate behavioral output and thereby give rise to fundamental innate behavioral states.