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, 594 (10), 2607-28

The Incretin Hormone Glucagon-Like Peptide 1 Increases Mitral Cell Excitability by Decreasing Conductance of a Voltage-Dependent Potassium Channel


The Incretin Hormone Glucagon-Like Peptide 1 Increases Mitral Cell Excitability by Decreasing Conductance of a Voltage-Dependent Potassium Channel

Nicolas Thiebaud et al. J Physiol.


Key points: The gut hormone called glucagon-like peptide 1 (GLP-1) is a strong moderator of energy homeostasis and communication between the peripheral organs and the brain. GLP-1 signalling occurs in the brain; using a newly developed genetic reporter line of mice, we have discovered GLP-synthesizing cells in the olfactory bulb. GLP-1 increases the firing frequency of neurons (mitral cells) that encode olfactory information by decreasing activity of voltage-dependent K channels (Kv1.3). Modifying GLP-1 levels, either therapeutically or following the ingestion of food, could alter the excitability of neurons in the olfactory bulb in a nutrition or energy state-dependent manner to influence olfactory detection or metabolic sensing. The results of the present study uncover a new function for an olfactory bulb neuron (deep short axon cells, Cajal cells) that could be capable of modifying mitral cell activity through the release of GLP-1. This might be of relevance for the action of GLP-1 mimetics now widely used in the treatment of diabetes.

Abstract: The olfactory system is intricately linked with the endocrine system where it may serve as a detector of the internal metabolic state or energy homeostasis in addition to its classical function as a sensor of external olfactory information. The recent development of transgenic mGLU-yellow fluorescent protein mice that express a genetic reporter under the control of the preproglucagon reporter suggested the presence of the gut hormone, glucagon-like peptide (GLP-1), in deep short axon cells (Cajal cells) of the olfactory bulb and its neuromodulatory effect on mitral cell (MC) first-order neurons. A MC target for the peptide was determined using GLP-1 receptor binding assays, immunocytochemistry for the receptor and injection of fluorescence-labelled GLP-1 analogue exendin-4. Using patch clamp recording of olfactory bulb slices in the whole-cell configuration, we report that GLP-1 and its stable analogue exendin-4 increase the action potential firing frequency of MCs by decreasing the interburst interval rather than modifying the action potential shape, train length or interspike interval. GLP-1 decreases Kv1.3 channel contribution to outward currents in voltage clamp recordings as determined by pharmacological blockade of Kv1.3 or utilizing mice with Kv1.3 gene-targeted deletion as a negative control. Because fluctuations in GLP-1 concentrations monitored by the olfactory bulb can modify the firing frequency of MCs, olfactory coding could change depending upon nutritional or physiological state. As a regulator of neuronal activity, GLP-1 or its analogue may comprise a new metabolic factor with a potential therapeutic target in the olfactory bulb (i.e. via intranasal delivery) for controlling an imbalance in energy homeostasis.


Figure 1
Figure 1. Preproglucagon positive (PPG+) short axon cells are visualized in the OB
A, photomicrograph of a representative coronal section of the mouse OB of a PPG‐YFP mouse demonstrating native YFP labelling of dSAC within the GCL. Also visible in the section are other neurolamina, MCL, EPL and the glomerular cell layer (GLM). Note the position of the soma in the upper portion of the GCL and the axon projections into the MCL and EPL. B, same as in (A) but labelled using a metal‐intensified diaminobenzidine reaction in conjunction with an anti‐GFP antibody to optimally visualize the short axon morphology. Inset: higher magnification of the boxed area is shown on the right. Note that the YFP‐immunoreactive cells are stellate with dendrites containing spine‐like structures or a varicose appearance.
Figure 2
Figure 2. GLP‐1R is expressed in the granular cell and MCLs of the mouse OB
A, left: photomicrographs of representative coronal sections of the OB of an OMP‐GFP mouse carrying a transgene for olfactory marker protein, OMP. The six‐panel composite demonstrates fluorescence labelling using an antibody directed against GLP‐1R (left; red) with no signal in the GLM, defined labelling in the MCL and scattered labelling in the GCL. Merged image on the right using double‐colour fluorescence strategy to visualize the GFP (green) and receptor (red) overlay. DAPI nuclear stain (blue). Entire OB for perspective shown on the right. B, RT‐PCR agarose electrophoresis gel using whole OB tissue as the template yields the anticipated size product (452 bp) for the GLP‐1R. C, representative photomicrograph composites as in (A) where a peptide binding assay was performed to visualize (top) GLP‐1 biotin conjugate binding (GLP‐1‐biotin) competing with combined (bottom) GLP‐1‐biotin plus GLP‐1 unconjugated binding (GLP‐1 cold). D, representative photomicrograph composite in which the section was co‐labelled with anti‐GLP‐1R (red, top), anti‐IR kinase (green, middle) with the merged image indicating MCs that putatively exhibit both co‐labelled proteins (yellow, bottom). DAPI nuclear stain (blue) is cropped below.
Figure 3
Figure 3. MCs internalize of fluorescent Ex4
Photomicrograph composites of OB sections from Thy1‐YFP mice injected with fluorescent Ex4 (Ex4‐647) showing intracellular uptake of Ex4 in the MCs (top). GLP‐1R−/− mice do not show uptake of Ex4‐647 in the MCL (bottom). DAPI, nuclear stain (blue); GLP‐1R−/–, GLP‐1R‐null mice; Merge, merged image on the right using double‐colour fluorescence strategy to visualize the YFP (green) or DAPI (blue) with Ex4 (purple) overlay.
Figure 4
Figure 4. GLP‐1 increases MC firing frequency via shortening the spike interburst interval
A, line graph of AP firing frequency (event frequency) and associated raster plot for a representative MC in response to bath application of 1 μm GLP‐1 to the slice. Dashed line and black bar, application of GLP‐1 proceeded by control artificial cerebral spinal fluid (Control ACSF) to establish baseline firing frequency and followed by an aCSF wash interval (Wash ACSF). B, enlarged resolution of AP firing pattern sampled at points a, b and c from (A). C, left: line graph of the AP firing frequency for the individual MCs sampled as in (A) and (right) bar graph of the mean firing frequency normalized to that of initial control ACSF condition. D, same as (C) but for 100 nm GLP‐1. E, bar graphs of the (left) ISI and (right) interburst interval for the AP activity recorded for the population of GLP‐1 responsive MCs in (C). F, same as (E) but for the AP activity recorded for the population of GLP‐1 responsive MCs in (D). DF, different letters indicate significantly‐different means; non‐parametric repeated measure one‐way analysis of variance (ANOVA; Friedman test) followed by a Bonferoni post hoc comparison. P values are as indicated on the graphs.
Figure 5
Figure 5. GLP‐1 analogue, Ex4, increases MC firing frequency via shortening the spike interburst interval
A and B, same experimental paradigm as in Fig. 4 A and B but for bath application of 1 μm Ex4. CD, same spike analysis, statistical metric and notations as in Fig. 4 C and E in the analysis of Ex4 as opposed to natural peptide GLP‐1.
Figure 6
Figure 6. GLP‐1 decreases the excitation threshold for MC firing
A, representative APs recorded in a representative MC held near resting potential and injected with a family of 500 ms long currents in 25 pA increments under control (Control) and following bath application of 1 μm GLP‐1 to the slice (GLP‐1). B, line graph of the mean AP firing frequency for a population of MCs (n = 6) recorded as in (A) plotted against injected current. **Significantly different means; repeated measure two‐way ANOVA; Bonferoni's post hoc test, using hormone treatment as the factor. **P ≤ 0.01, ****P ≤ 0.0001.
Figure 7
Figure 7. The threshold for activation of the GLP‐1 sensitive current shifts with a change in the equilibrium potential for K+ (EK) generated by altering the external concentration for K+
A, representative I/C plotted relationship of currents evoked using a 400 ms voltage ramp from –120 to +40 mV before (a, Control, black line) and after (b, GLP‐1, grey line) bath application of 1 μm GLP‐1 to the slice under conditions of 2.5 mm (left) vs. 8.5 mm (right) external KCl concentration. B, the GLP‐1 sensitive current is calculated by a subtraction of a – b to yield an activation threshold of –56.3 ± 1.5 mV using a 2.5 mm external K+ concentration (left) compared with the right‐shifted activation threshold of –46.7 ± 3.2 mV using a 8.5 mm external K+ concentration (right).
Figure 8
Figure 8. MGTX increases AP firing frequency in MCs by decreasing the interburst interval and not the ISI
A, representative APs recorded in a representative MC under (a) baseline ASCF control bath conditions (Control), (b) after 5 min of 1 nm MGTX and (c) after 15 min of 1 nm MGTX stimulation, reflecting the slow K on reported for this small peptide molecule that blocks the vestibule of the Kv1.3 channel. B, left: line graph of the mean firing frequency over time for five sampled MCs normalized to initial AP firing rate before MGTX treatment (time 0 min). Right: same population of MCs plotted to examine AP mean ISI (closed symbol) and interburst interval (open symbols) over time.
Figure 9
Figure 9. The magnitude of MC voltage‐activated outward currents are decreased following GLP‐1 application in WT mice but not for mice with a Kv1.3‐targeted deletion (Kv1.3–/–)
A, top: representative family of voltage‐activated currents elicited by stepping the command voltage (V c) in 10 mV increments (–100 to +40 mV) from a holding voltage (V h) of –80 mV using a 400 ms pulse duration (Pd) and a 45 s interpulse interval. MC recordings were acquired from WT mice. TTX was applied to the bath to isolate outward potassium conductances (Control, 1 nm TTX) before (left traces) and after (right traces) bath application of the peptide (+ 1 μm GLP‐1). Bottom left: plotted I/C relationship for five MCs recorded as in (A). Solid symbols, before (Control); open symbols, after GLP‐1(GLP‐1 1 μm) bath application. Significantly different means; repeated measures two‐way ANOVA; Bonfernoni's post hoc test, *P ≤ 0.05; **P ≤ 0.001. Right, same as left I/C but normalized to that of the +40 mV voltage step. B, as in (A), except MCs were recorded from Kv1.3−/− mice (Kv1.3−/−).
Figure 10
Figure 10. GLP‐1 fails to modulate MC firing frequency in mice with a Kv1.3‐targed deletion (Kv1.3−/−)
A and B, same experimental paradigm as in Fig. 3 A and B, although MC were acquired in slices from Kv1.3−/− mice. C, same spike analysis, statistical metric and notations as in Fig. 3 C, as acquired from Kv1.3−/− rather than WT mice.
Figure 11
Figure 11. Schematic of putative roles for GLP‐1 in the OB
Glucagon‐like peptide‐1 (GLP‐1) signalling pathways act to increase MC excitability, where several hypotheses are proposed for its role in the OB. (1) GLP‐1 could be used locally for dSAC‐to‐MC transmission or combined neurotransmission/neuromodulation of MC activity; (2) MCs could sense GLP‐1 as a metabolic signal of nutritional state derived from peripheral release in the gut and brought to the OB through the circulation; (3) centrifugal input could provide metabolic control of dSACs; or (4) hormones driven by peripheral metabolic signals may govern dSAC peptidergic transmission. Regardless of the route of GLP‐1 generation, its inducibility or regulation of release, our data demonstrate that a downstream target is the Kv1.3 ion channel. The Kv1.3 channel (brown) is predominantly expressed on MCs where it is known to be a substrate for IR kinase (blue) phosphorylation on the N‐ and C‐terminal aspects of the channel protein (Y111‐113, Y137 and Y479) (Fadool & Levitan, 1998; Fadool et al. 2000). Insulin‐induced phosphorylation of Kv1.3 (inhibitory line) decreases Kv current magnitude by decreasing the Pr open of the channel rather than altering its unitary conductance (Fadool et al. 2000). GLP‐1 is known to stimulate insulin release in pancreatic β cells, a process that is glucose‐dependent, although its activation of IR kinase signalling in the brain is not known (arrow). Because Kv1.3 is part of a scaffold of protein–protein interactions (Marks & Fadool, 2007), it is highly probable that IR kinase (Fadool et al. 2000; Fadool et al. 2011), glucose signalling (Tucker et al. 2013) and GLP‐1 signalling converge at the level of this channel, although whether MCs homogeneously express all three metabolic factors that regulate their firing frequency is not fully understood. Deletion of the Kv1.3 channel results in a thin body type and resistance to diet and genetic obesity (Fadool et al. 2004; Tucker et al. 2008). Metabolic factors that decrease Kv1.3 ion channel activity increase the AP firing frequency in MCs that is considered to provide odour quality coding of olfactory information. Such modulation may in turn drive changes in odour‐dependent food seeking behaviours in an environment where glucose, insulin and GLP‐1 do not remain static and can also be perturbed with metabolic dysfunction or obesity. GCL, granule cell layer; MCL, mitral cell layer; GLM, glomerular layer.

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