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. 2010 Aug;59(8):1890-8.
doi: 10.2337/db10-0128. Epub 2010 Jun 3.

Leptin directly depolarizes preproglucagon neurons in the nucleus tractus solitarius: electrical properties of glucagon-like Peptide 1 neurons

Kazunari Hisadome  1 , Frank Reimann, Fiona M Gribble, Stefan Trapp
Affiliations

Leptin directly depolarizes preproglucagon neurons in the nucleus tractus solitarius: electrical properties of glucagon-like Peptide 1 neurons

Kazunari Hisadome et al. Diabetes. 2010 Aug.

Abstract

Objective: Glucagon-like peptide (GLP)-1 inhibits food intake, acting both in the periphery and within the central nervous system. It is unclear if gut-derived GLP-1 can enter the brain, or whether GLP-1 from preproglucagon (PPG) cells in the lower brainstem is required to activate central GLP-1 receptors. Brainstem PPG neurons, however, have been poorly characterized, due to the difficulties in identifying these cells while viable. This study provides data on the electrical properties of brainstem PPG cells and their regulation by orexigenic and anorexigenic peptides.

Research design and methods: Transgenic mice expressing Venus under control of the PPG promoter were used to identify PPG neurons in vitro in brainstem slice preparations for electrophysiological recordings. RESULTS The majority of PPG neurons were spontaneously active. Further electrical and molecular characterization revealed that GLP-1 receptor activation had no pre- or postsynaptic effect and that PPG neurons lack GLP-1 receptors. Similarly, they were unresponsive to PYY and ghrelin. In contrast, leptin rapidly and reversibly depolarized these neurons. Responses to electrical stimulation of the solitary tract suggest that PPG cells are mostly second-order neurons, receiving direct input from vagal afferent fibers. Both evoked and spontaneous excitatory postsynaptic currents were predominantly glutamatergic.

Conclusions: The study introduces PPG-promoter-Venus transgenic mice as a viable and important tool to study brainstem PPG cells. PPG neuron activity is directly modulated by leptin but was unaffected by other satiety or hunger peptides. Direct synaptic input from the solitary tract suggests that peripheral signals (including GLP-1) could modulate PPG cells via vagal afferents.

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Figures

FIG. 1.
FIG. 1.
Basic properties of PPG neurons of the NTS. A: Photomicrograph showing the location of PPG-Venus neurons (green fluorescence) in relation to the cholinergic (red fluorescence) dorsal vagal nucleus (DMNX) and hypoglossal nucleus (HN) in a 30-μm coronal brainstem slice at position Bregma −7.75 mm. The central canal and the dorsal border of the brainstem are indicated by dotted lines. B: Horizontal brainstem slice demonstrating the catecholaminergic cells (red fluorescence signifying anti-TH immunoreactivity) and PPG cells (green fluorescence) are two completely distinct cell populations that occupy overlapping areas of the NTS. The dotted line indicates the midline. CC, central canal. Confocal analysis of selected regions from these slices suggests that there is no direct contact between TH-positive and PPG cells (data not shown). C: Individual PPG neurons were identified by Venus fluorescence (top, patch pipette position indicated by dotted lines) and recordings were established under DIC illumination (bottom). D: In the cell-attached configuration while waiting for perforation, the firing phenotype of the neuron was observed: i, regular firing; ii, silent; iii, burst firing. E: Typical perforated-patch current-clamp recordings from regular firing neuron (i) and burst firing cell (ii). The fluctuations in membrane potential underlying the burst firing behavior persisted in TTX. F: injection of a −40 pA current pulse for 200 ms elicited an A-type K+ current (arrow) upon termination of the pulse in all three cell types. (A high-quality digital representation of this figure is available in the online issue.)
FIG. 2.
FIG. 2.
PPG neurons have postsynaptic ionotropic glutamate receptors. A: Voltage-clamp recording at a holding potential of −70 mV demonstrating spontaneous excitatory postsynaptic current (EPSC) activity and its inhibition by the glutamate receptor antagonist kynurenic acid (Kyn; 1 mmol/l). B: Mean data from recordings as shown in A and from experiments with the non-NMDA receptor antagonist DNQX (10 μmol/l). Mean EPSC frequency was determined over 3 min under each condition. **P < 0.01 against control. C: Bath application of 0.1 mmol/l glutamate during a perforated patch clamp recording caused a reversible depolarization by ∼40 mV in this PPG neuron. D: Mean effects of glutamate and glutamate plus DNQX (20 μmol/l) on the input resistance (RM) and membrane potential (EM) of PPG neurons. Note that DNQX almost completely abolishes the glutamate effect. **P < 0.01 against control; ##P < 0.01 against glutamate. Numbers of cells tested (n) are given above the bars.
FIG. 3.
FIG. 3.
Stimulation of the solitary tract evokes glutamate EPSCs in PPG neurons. A: Voltage clamp recording from a PPG neuron in a horizontal slice at a holding potential of −70 mV. Repetitive stimulation of the solitary tract (five pulses at 50 Hz every 20 s) evoked EPSCs of progressively smaller amplitude, small failure rate, and low jitter (i.e., mono-synaptic) in most of these cells. A total of 10 consecutive traces are overlaid. B: Some cells showed higher failure rates and larger jitter. A total of 10 consecutive traces are overlaid. C: All cells tested showed a marked reduction in EPSC amplitude in the presence of glutamate receptor antagonists kynurenic acid (kyn) or DNQX (gray trace in i and ii). D: Mean data from recordings as shown in C. **P < 0.01 against control. Numbers of cells tested (n) are given above the bars.
FIG. 4.
FIG. 4.
PPG neurons are not sensitive to gut peptides GLP-1 and PYY. A: Perforated patch clamp recording in current clamp demonstrating the lack of effect of 100 nmol/l GLP-1 on PPG cell activity. The top trace shows the instantaneous firing frequency over the course of the recording, and the bottom traces show brief periods of the original recording at time points indicated by arrows. B: Mean firing frequency in the absence and presence of 100 nmol/l GLP-1 or 100 nmol/l of the GLP-1 receptor agonist exendin-4. C: Typical single-cell RT-PCR analysis for PPG and the GLP-1 receptor (GLP-1R) for three PPG neurons and controls. The 2% agarose gel demonstrating that the 186-bp PCR product for PPG and the 292-bp PCR product for GLP-1R can be obtained from brainstem cDNA (BS 1:1,000 dilution; positive control) with the primers specified in Table 1. In contrast, cytoplasm extracted from single cells showing Venus fluorescence (–3) was only positive for PPG, but not GLP-1R. PIP, pipette solution without cytoplasm extracted from cell (negative control). D: Neither exendin-4 nor the gut peptide PYY had any effect on spontaneous EPSCs in PPG neurons. Left: Typical voltage-clamp recordings in the absence or presence of exendin-4. Right: Typical recordings in the absence or presence of PYY. E: Mean data from recordings as shown in B for frequency and amplitude of spontaneous EPSCs. F: The peptide ghrelin (100 nmol/l), released from stomach as orexinergic signal, had no effect on membrane potential (EM), input resistance (RM), or action potential frequency. Numbers of cells tested (n) are given above the bars.
FIG. 5.
FIG. 5.
Leptin depolarizes PPG neurons. A: Response to local application of 20 nmol/l leptin. Top trace shows the action potential firing rate (FR), and the bottom trace shows the full current-clamp recording. B: Periods of the recording indicated in A by a, b, and c at an extended timescale. Leptin caused a depolarization of the membrane potential (EM), decrease in membrane resistance (RM), and increase in action potential firing rate (FR). C: Mean data from eight cells showing the peak response for each parameter. *P < 0.05 against control; **P < 0.01 against control. Numbers of cells tested (n) are given above the bars.
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