Probes for INS

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide-1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. As GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized (OVX) mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased following a 48 h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin (9-39) in ad libitum fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

Significance Statement Reproductive dysfunction is associated with metabolic imbalance, and identifying the underlying molecular mechanisms linking metabolic status with reproductive function is of great importance. Kisspeptin neurons (Kiss1) located in the arcuate nucleus of the hypothalamus (ARC) are essential for fertility and are potently inhibited during negative energy balance; this inhibition occurs in the presence or absence of ovarian steroids. Preproglucagon-expressing neurons located in the brainstem send abundant fiber projections to the ARC where they release the anorexigenic neuropeptide, glucagon-like peptide-1 (GLP-1). The aim of these studies was to determine the interaction of the CNS GLP-1 system with ARC Kiss1 activity to potentially provide a link between systems that control energy balance with those that control reproductive neuroendocrine output.

Abstract

Objective

The prevalence of obesity and related co-morbidities is reaching pandemic proportions. Today, the most effective obesity treatments are glucagon-like peptide 1 (GLP-1) analogs and bariatric surgery. Interestingly, both intervention paradigms have been associated with adaptive growth responses in the gut; however, intestinotrophic mechanisms associated with or secondary to medical or surgical obesity therapies are poorly understood. Therefore, the objective of this study was to assess the local basal endogenous and pharmacological intestinotrophic effects of glucagon-like peptides and bariatric surgery in mice.

Methods

We used in situ hybridization to provide a detailed and comparative anatomical map of the local distribution of GLP-1 receptor (Glp1r), GLP-2 receptor (Glp2r), and preproglucagon (Gcg) mRNA expression throughout the mouse gastrointestinal tract. Gut development in GLP-1R-, GLP-2R-, or GCG-deficient mice was compared to their corresponding wild-type controls, and intestinotrophic effects of GLP-1 and GLP-2 analogs were assessed in wild-type mice. Lastly, gut volume was determined in a mouse model of vertical sleeve gastrectomy (VSG).

Results

Comparison of Glp1r, Glp2r, and Gcg mRNA expression indicated a widespread, but distinct, distribution of these three transcripts throughout all compartments of the mouse gastrointestinal tract. While mice null for Glp1r or Gcg showed normal intestinal morphology, Glp2r^−/− mice exhibited a slight reduction in small intestinal mucosa volume. Pharmacological treatment with GLP-1 and GLP-2 analogs significantly increased gut volume. In contrast, VSG surgery had no effect on intestinal morphology.

Conclusion

The present study indicates that the endogenous preproglucagon system, exemplified by the entire GCG gene and the receptors for GLP-1 and GLP-2, does not play a major role in normal gut development in the mouse. Furthermore, elevation in local intestinal and circulating levels of GLP-1 and GLP-2 achieved after VSG has limited impact on intestinal morphometry. Hence, although exogenous treatment with GLP-1 and GLP-2 analogs enhances gut growth, the contributions of endogenously-secreted GLP-1 and GLP-2 to gut growth may be more modest and highly context-dependent.

Glutamatergic neurons that express pre-proglucagon (PPG) and are immunopositive (+) for glucagon-like peptide-1 (i.e., GLP-1+ neurons) are located within the caudal nucleus of the solitary tract (cNTS) and medullary reticular formation in rats and mice. GLP-1 neurons give rise to an extensive central network in which GLP-1 receptor (R) signaling suppresses food intake, attenuates rewarding, increases avoidance, and stimulates stress responses, partly via . GLP-1R signaling within the cNTS. In mice, noradrenergic (A2) cNTS neurons express GLP-1R, whereas PPG neurons do not. In the present study, confocal microscopy in rats confirmed that prolactin-releasing peptide (PrRP)+ A2 neurons are closely apposed by GLP-1+ axonal varicosities. Surprisingly, GLP-1+ appositions were also observed on dendrites of PPG/GLP-1+ neurons in both species, and electron microscopy in rats revealed that GLP-1+ boutons form asymmetric synaptic contacts with GLP-1+ dendrites. However, RNAscope confirmed that rat GLP-1 neurons do not express GLP-1R mRNA. Similarly, Ca2+ imaging of somatic and dendritic responses in mouse ex vivo slices confirmed that PPG neurons do not respond directly to GLP-1, and a mouse cross-breeding strategy revealed that fewer than 1% of PPG neurons co-express GLP-1R. Collectively, these data suggest that GLP-1R signaling pathways modulate the activity of PrRP+ A2 neurons, and also reveal a local "feed-forward" synaptic network among GLP-1 neurons that apparently does not utilize GLP-1R signaling. This local GLP-1 network may instead use glutamatergic signaling to facilitate dynamic and potentially selective recruitment of GLP-1 neural populations that shape behavioral and physiological responses to internal and external challenges.

Place cells in the CA1 region of the hippocampus express location-specific firing despite receiving a steady barrage of heterogeneously tuned excitatory inputs that should compromise output dynamic range and timing. We examined the role of synaptic inhibition in countering the deleterious effects of off-target excitation. Intracellular recordings in behaving mice demonstrate that bimodal excitation drives place cells, while unimodal excitation drives weaker or no spatial tuning in interneurons. Optogenetic hyperpolarization of interneurons had spatially uniform effects on place cell membrane potential dynamics, substantially reducing spatial selectivity. These data and a computational model suggest that spatially uniform inhibitory conductance enhances rate coding in place cells by suppressing out-of-field excitation and by limiting dendritic amplification. Similarly, we observed that inhibitory suppression of phasic noise generated by out-of-field excitation enhances temporal coding by expanding the range of theta phase precession. Thus, spatially uniform inhibition allows proficient and flexible coding in hippocampal CA1 by suppressing heterogeneously tuned excitation.

Activity-dependent transcriptional responses shape cortical function. However, a comprehensive understanding of the diversity of these responses across the full range of cortical cell types, and how these changes contribute to neuronal plasticity and disease, is lacking. To investigate the breadth of transcriptional changes that occur across cell types in the mouse visual cortex after exposure to light, we applied high-throughput single-cell RNA sequencing. We identified significant and divergent transcriptional responses to stimulation in each of the 30 cell types characterized, thus revealing 611 stimulus-responsive genes. Excitatory pyramidal neurons exhibited inter- and intralaminar heterogeneity in the induction of stimulus-responsive genes. Non-neuronal cells showed clear transcriptional responses that may regulate experience-dependent changes in neurovascular coupling and myelination. Together, these results reveal the dynamic landscape of the stimulus-dependent transcriptional changes occurring across cell types in the visual cortex; these changes are probably critical for cortical function and may be sites of deregulation in developmental brain disorders.