Probes for INS

The median preoptic nucleus (MnPO) is a putative integrative region that contributes to body fluid balance. Activation of the MnPO can influence thirst but it is not clear how these responses are linked to body fluid homeostasis. We used Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to determine the role of the MnPO in drinking behavior and vasopressin release in response to peripheral angiotensin II (ANG II) or 3% hypertonic saline in adult male Sprague-Dawley rats (250-300g). Rats were anesthetized with isoflurane and stereotaxically injected with an inhibitory DREADD (rAAV5-CaMKIIa-hM4D(Gi)-mCherry) or control (rAAV5-CaMKIIa-mCherry) virus in the MnPO. After 2 weeks’ recovery, a subset of rats were used for extracellular recordings to verify functional effects of ANG II or hyperosmotic challenges in MnPO slice preparations. Remaining rats were used in drinking behavior studies. Each rat was administered either 10mg/kg of exogenous clozapine-N-oxide (CNO) to inhibit DREADD-expressing cells or vehicle ip followed by a test treatment with either 2mg/kg ANG II or 3% hypertonic saline (1mL/100g bw) sc, twice per week for two separate treatment weeks. CNO-induced inhibition during either test treatment significantly attenuated drinking responses compared to vehicle treatments and controls. Brain tissue processed for cFos immunohistochemistry showed decreased expression with CNO-induced inhibition during either test treatment in the MnPO and downstream nuclei compared to controls. CNO-mediated inhibition significantly attenuated treatment-induced increases in plasma vasopressin compared to controls. The results indicate inhibition of CaMKIIa-expressing MnPO neurons significantly reduces drinking and vasopressin release in response to ANG II or hyperosmotic challenge.

Significance Statement The MnPO is an important regulatory center that influences thirst, cardiovascular and neuroendocrine function. Activation of different MnPO neuronal populations can inhibit or stimulate water intake. However, the role of the MnPO and its pathway-specific projections during ANG II and hyperosmotic challenges still have not yet been fully elucidated. These studies directly address this by using DREADDs to acutely and selectively inhibit pathway-specific MnPO neurons, and uses techniques that measure changes at the protein, neuronal, and overall physiological and behavioral level. More importantly, we have been able to demonstrate that physiological challenges related to extracellular (ANG II) or cellular (hypertonic saline) dehydration activate MnPO neurons that may project to different parts of the hypothalamus.

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.

Basal cell carcinoma (BCC) is the most frequent cancer in humans and results from constitutive activation of the Hedgehog pathway1. Several Smoothened inhibitors are used to treat Hedgehog-mediated malignancies, including BCC and medulloblastoma2. Vismodegib, a Smoothened inhibitor, leads to BCC shrinkage in the majority of patients with BCC3, but the mechanism by which it mediates BCC regression is unknown. Here we used two genetically engineered mouse models of BCC4 to investigate the mechanisms by which inhibition of Smoothened mediates tumour regression. We found that vismodegib mediates BCC regression by inhibiting a hair follicle-like fate and promoting the differentiation of tumour cells. However, a small population of tumour cells persists and is responsible for tumour relapse following treatment discontinuation, mimicking the situation found in humans5. In both mouse and human BCC, this persisting, slow-cycling tumour population expresses LGR5 and is characterized by active Wnt signalling. Combining Lgr5 lineage ablation or inhibition of Wnt signalling with vismodegib treatment leads to eradication of BCC. Our results show that vismodegib induces tumour regression by promoting tumour differentiation, and demonstrates that the synergy between Wnt and Smoothened inhibitors is a clinically relevant strategy for overcoming tumour relapse in BCC.