A hindbrain inhibitory microcircuit mediates vagally-coordinated glucose regulation

Figure 1. Cre-dependent expression and response to activation of designer receptor exclusively activated by designer drugs (DREADDs) in dorsal hindbrain GABAergic neurons. (a) Expression of excitatory DREADDs was induced though a stereotaxic microinjection of pAAV8-hSyn-DIO-hM3Dq into the dorsal vagal complex (DVC) of a vGAT-Cre mouse. (b) Light microscopic images of endogenous mCherry expression in the DVC. Left: low magnification image of the DVC. Right: higher magnification centered on the nucleus of the solitary tract (NTS) three weeks after AAV inoculation. The dorsal motor nucleus of the vagus (X) is encircled. Expression was prominent in the NTS, but also could include a few GABAergic interneurons located in the DMV, which have been described previously56. (c) Illustration of the recording configuration, with the recording pipette in the NTS. Image under combined fluorescent and IR/DIC illumination during a recording from an mCherry expressing NTS neuron is to the right of the diagram. Recording pipette is visible attached to an mCherry expressing neuron. Lower. Images of an NTS neuron that was filled with biocytin during a recording and was visualized post hoc with AMCA (left); the…

Figure 2. Activation of GABAergic neurons in the DVC inhibits activity of neurons in the dorsal motor nucleus of the vagus (DMV). (a) Top: Illustration of the synaptic pathway tested. A GABA neuron in the NTS with a synaptic connection to a motor neuron in the DMV (X) is illustrated. Bottom: Illustration indicating the location of recordings in the DMV. (b) Post hoc confirmation of the location of a recorded and biocytin-filled DMV neuron. No co-localization with mCherry was observed in DMV neurons. (c) Top: Spontaneous inhibitory postsynaptic currents (sIPSCs) in a DMV neuron before and after CNO application. Bottom: Mean sIPSC frequency and amplitude (n = 6). CNO significantly increased sIPSC frequency (p < 0.05), but not amplitude. (d) Top: Action potential firing in a DMV neuron before and after CNO application. Bottom: Pie graph illustrating the proportion of AP firing responses in DMV neurons to bath application of CNO (left) and mean action potential (AP) frequency in the neurons in which a decrease in AP frequency was induced by CNO (n = 5; p < 0.05; right). (e) Mean blood glucose concentration after in vivo systemic administration of CNO (1 mg/kg) or vehicle (0.9% saline + 0.5%…

Figure 3. Peripheral pathway mediating CNO-induced hyperglycemia. (a) Illustration of hypothesized pathway involved in the elevation of blood glucose after remote activation of GABAergic hindbrain neurons. The inhibitory synaptic connection from NTS neurons to the DMV is shown, as is the increase in glucagon release from the pancreas. Increased hepatic gluconeogenesis could result from decreased vagal input (shown) and/or glucagon actions in the liver. (b) Change in blood glucose after 90 mins of vehicle, CNO (1 mg/kg), or glucose (0.26 mg/kg) injection (i.p.). (c) Mean serum glucagon (left) and insulin (right) concentration 90 min post-treatment. (d) Mean expression (fold change) of hepatic PEPCK1 mRNA expression (normalized to β-actin expression). (e) Western blots indicated that pAKT/AKT and pLDHA/LDHA protein expression in gastrocnemius muscle were unaltered at the same time point. For each protein, example blots were cropped from the same gel, with boxes and lines added for clarity; exposures are unaltered. The full-length blots are shown in Supplemental Fig. S4.

Figure 4. Activation of the inhibitory hM4Di DREADD in GABAergic hindbrain neurons does not alter blood glucose concentration or DMV neuron activity. (a) Diagram showing recording location in the NTS and representative traces showing action potentials in an mCherry expressing NTS neuron before (left) and after CNO (right; 10 μM) application in a mouse that received the hM4Di viral construct. (b) Mean AP firing and membrane potential before and after CNO application (n = 8). *Significant difference from nACSF (paired t-test; p < 0.05). (c) Diagram showing recording location in the DMV (left). Pie graph (right) illustrating AP firing responses in NTS and DMV neurons. AP firing was decreased by CNO in NTS neurons but was unchanged in most DMV neurons (n = 5). (d) Blood glucose concentration after in vivo administration of CNO (1 mg/kg) or vehicle (0.9% saline + 0.5% DMSO). Asterisks indicate significant change from time 0, but no differences between treatment groups.