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CCK stimulation of GLP-1 neurons involves α1-adrenoceptor-mediated increase in glutamatergic synaptic inputs.

Hisadome K, Reimann F, Gribble FM, Trapp S - Diabetes (2011)

Bottom Line: Inhibition of adrenergic signaling abolished the excitatory action of CCK.CCK activates NTS-PPG cells by a circuit involving adrenergic and glutamatergic neurons.NTS-PPG neurons integrate a variety of peripheral signals that indicate both long-term energy balance and short-term nutritional and digestional status to produce an output signal to feeding and autonomic circuits.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, London, UK.

ABSTRACT

Objective: Glucagon-like peptide 1 (GLP-1) is involved in the central regulation of food intake. It is produced within the brain by preproglucagon (PPG) neurons, which are located primarily within the brain stem. These neurons project widely throughout the brain, including to the appetite centers in the hypothalamus, and are believed to convey signals related to satiety. Previous work demonstrated that they are directly activated by leptin and electrical activity of the afferent vagus. Another satiety hormone, cholecystokinin (CCK), has also been linked to activation of brain stem neurons, suggesting that it might act partially via centrally projecting neurons from the nucleus tractus solitarius (NTS). The aim of this study was to investigate the neuronal circuitry linking CCK to the population of NTS-PPG neurons.

Research design and methods: Transgenic mice expressing yellow fluorescent protein (Venus) under the control of the PPG promoter were used to identify PPG neurons in vitro and to record their electrical and pharmacological profile.

Results: PPG neurons in the NTS were excited by CCK and epinephrine, but not by the melanocortin receptor agonist melanotan II. Both CCK and epinephrine acted to increase glutamatergic transmission to the PPG neurons, and this involved activation of α(1)-adrenergic receptors. Inhibition of adrenergic signaling abolished the excitatory action of CCK.

Conclusions: CCK activates NTS-PPG cells by a circuit involving adrenergic and glutamatergic neurons. NTS-PPG neurons integrate a variety of peripheral signals that indicate both long-term energy balance and short-term nutritional and digestional status to produce an output signal to feeding and autonomic circuits.

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Related in: MedlinePlus

CCK increases spontaneous activity of PPG neurons. A: Current-clamp recording demonstrating that bath application of 100 nmol/L CCK-8s led to an increase in spontaneous action potential firing frequency of this PPG neuron. B: A plot of the firing frequency for the recording shown in A (the part of the recording shown in A is indicated by gray background). C: Short segments of the original current-clamp recording from B at time points indicated by i, ii, iii, and iv. D: Mean data for firing frequency from experiments as depicted in A and B. CCK-8 (100 nmol/L) significantly increased firing rate. This effect of CCK-8s was occluded in the presence of the non-NMDA glutamate receptor antagonist DNQX. Number of recordings for each condition is given above the bars. *P < 0.05. E: Typical single-cell RT-PCR analysis for PPG and the CCK receptors (CCKAR, CCKBR) for three PPG neurons and controls. Agarose gel (2%) demonstrating that the 186-bp PCR product for PPG, the 285-bp PCR product for CCKAR, and the 341-bp product for CCKBR can be obtained from brain stem cDNA (1:100 dilution; positive control; indicated by arrows) with the primers specified in Table 1. In contrast, cytoplasm extracted from single cells showing eYFP fluorescence (cell1, cell2, cell3) was only positive for PPG, but not CCKAR or CCKBR (only bands for primers visible). Negative (neg) control: pipette solution without cytoplasm extracted from cell. Molecular weight ladder shows bands at 100-bp intervals.
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Figure 1: CCK increases spontaneous activity of PPG neurons. A: Current-clamp recording demonstrating that bath application of 100 nmol/L CCK-8s led to an increase in spontaneous action potential firing frequency of this PPG neuron. B: A plot of the firing frequency for the recording shown in A (the part of the recording shown in A is indicated by gray background). C: Short segments of the original current-clamp recording from B at time points indicated by i, ii, iii, and iv. D: Mean data for firing frequency from experiments as depicted in A and B. CCK-8 (100 nmol/L) significantly increased firing rate. This effect of CCK-8s was occluded in the presence of the non-NMDA glutamate receptor antagonist DNQX. Number of recordings for each condition is given above the bars. *P < 0.05. E: Typical single-cell RT-PCR analysis for PPG and the CCK receptors (CCKAR, CCKBR) for three PPG neurons and controls. Agarose gel (2%) demonstrating that the 186-bp PCR product for PPG, the 285-bp PCR product for CCKAR, and the 341-bp product for CCKBR can be obtained from brain stem cDNA (1:100 dilution; positive control; indicated by arrows) with the primers specified in Table 1. In contrast, cytoplasm extracted from single cells showing eYFP fluorescence (cell1, cell2, cell3) was only positive for PPG, but not CCKAR or CCKBR (only bands for primers visible). Negative (neg) control: pipette solution without cytoplasm extracted from cell. Molecular weight ladder shows bands at 100-bp intervals.

Mentions: PPG neurons in the NTS were identified by their YFP fluorescence, and electrophysiological recordings were established under differential interference contrast optics as described previously (19). All experiments for this study were performed on the spontaneously active population of PPG neurons only. Any burst-firing cells were discarded (19). In current clamp (perforated-patch configuration) spontaneously active cells had a resting potential of −47 ± 1 mV (n = 29) and fired action potentials at a frequency of 1.5 ± 0.2 Hz (n = 28). Bath application of 100 nmol/L CCK octapeptide, sulfated (CCK-8s), had no significant effect on the resting membrane potential or the input resistance (data not shown) but led to a reversible increase in action potential frequency by 104 ± 34%, in 7 of 15 cells tested (Fig. 1), whereas no changes were observed in the remaining cells. This CCK-8s–induced increase in firing rate was abolished by bath application of 20 μmol/L DNQX, a non-N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, in 5 out of 5 cells tested, suggesting that CCK-8s activated PPG cells via an increase in glutamatergic synaptic activity, rather than directly by activation of postsynaptic CCK receptors on PPG neurons. In agreement with these results, single-cell RT-PCR analysis demonstrated that m-RNA for the CCK receptors CCKAR and CCKBR is absent from PPG neurons (n = 8) but present in cDNA from mouse brain stem (Fig. 1E). These results indicated the presence of central CCK receptors, which might reside on either vagal afferent terminals or another cell population within the brain stem, but not on PPG neurons.


CCK stimulation of GLP-1 neurons involves α1-adrenoceptor-mediated increase in glutamatergic synaptic inputs.

Hisadome K, Reimann F, Gribble FM, Trapp S - Diabetes (2011)

CCK increases spontaneous activity of PPG neurons. A: Current-clamp recording demonstrating that bath application of 100 nmol/L CCK-8s led to an increase in spontaneous action potential firing frequency of this PPG neuron. B: A plot of the firing frequency for the recording shown in A (the part of the recording shown in A is indicated by gray background). C: Short segments of the original current-clamp recording from B at time points indicated by i, ii, iii, and iv. D: Mean data for firing frequency from experiments as depicted in A and B. CCK-8 (100 nmol/L) significantly increased firing rate. This effect of CCK-8s was occluded in the presence of the non-NMDA glutamate receptor antagonist DNQX. Number of recordings for each condition is given above the bars. *P < 0.05. E: Typical single-cell RT-PCR analysis for PPG and the CCK receptors (CCKAR, CCKBR) for three PPG neurons and controls. Agarose gel (2%) demonstrating that the 186-bp PCR product for PPG, the 285-bp PCR product for CCKAR, and the 341-bp product for CCKBR can be obtained from brain stem cDNA (1:100 dilution; positive control; indicated by arrows) with the primers specified in Table 1. In contrast, cytoplasm extracted from single cells showing eYFP fluorescence (cell1, cell2, cell3) was only positive for PPG, but not CCKAR or CCKBR (only bands for primers visible). Negative (neg) control: pipette solution without cytoplasm extracted from cell. Molecular weight ladder shows bands at 100-bp intervals.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3198097&req=5

Figure 1: CCK increases spontaneous activity of PPG neurons. A: Current-clamp recording demonstrating that bath application of 100 nmol/L CCK-8s led to an increase in spontaneous action potential firing frequency of this PPG neuron. B: A plot of the firing frequency for the recording shown in A (the part of the recording shown in A is indicated by gray background). C: Short segments of the original current-clamp recording from B at time points indicated by i, ii, iii, and iv. D: Mean data for firing frequency from experiments as depicted in A and B. CCK-8 (100 nmol/L) significantly increased firing rate. This effect of CCK-8s was occluded in the presence of the non-NMDA glutamate receptor antagonist DNQX. Number of recordings for each condition is given above the bars. *P < 0.05. E: Typical single-cell RT-PCR analysis for PPG and the CCK receptors (CCKAR, CCKBR) for three PPG neurons and controls. Agarose gel (2%) demonstrating that the 186-bp PCR product for PPG, the 285-bp PCR product for CCKAR, and the 341-bp product for CCKBR can be obtained from brain stem cDNA (1:100 dilution; positive control; indicated by arrows) with the primers specified in Table 1. In contrast, cytoplasm extracted from single cells showing eYFP fluorescence (cell1, cell2, cell3) was only positive for PPG, but not CCKAR or CCKBR (only bands for primers visible). Negative (neg) control: pipette solution without cytoplasm extracted from cell. Molecular weight ladder shows bands at 100-bp intervals.
Mentions: PPG neurons in the NTS were identified by their YFP fluorescence, and electrophysiological recordings were established under differential interference contrast optics as described previously (19). All experiments for this study were performed on the spontaneously active population of PPG neurons only. Any burst-firing cells were discarded (19). In current clamp (perforated-patch configuration) spontaneously active cells had a resting potential of −47 ± 1 mV (n = 29) and fired action potentials at a frequency of 1.5 ± 0.2 Hz (n = 28). Bath application of 100 nmol/L CCK octapeptide, sulfated (CCK-8s), had no significant effect on the resting membrane potential or the input resistance (data not shown) but led to a reversible increase in action potential frequency by 104 ± 34%, in 7 of 15 cells tested (Fig. 1), whereas no changes were observed in the remaining cells. This CCK-8s–induced increase in firing rate was abolished by bath application of 20 μmol/L DNQX, a non-N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, in 5 out of 5 cells tested, suggesting that CCK-8s activated PPG cells via an increase in glutamatergic synaptic activity, rather than directly by activation of postsynaptic CCK receptors on PPG neurons. In agreement with these results, single-cell RT-PCR analysis demonstrated that m-RNA for the CCK receptors CCKAR and CCKBR is absent from PPG neurons (n = 8) but present in cDNA from mouse brain stem (Fig. 1E). These results indicated the presence of central CCK receptors, which might reside on either vagal afferent terminals or another cell population within the brain stem, but not on PPG neurons.

Bottom Line: Inhibition of adrenergic signaling abolished the excitatory action of CCK.CCK activates NTS-PPG cells by a circuit involving adrenergic and glutamatergic neurons.NTS-PPG neurons integrate a variety of peripheral signals that indicate both long-term energy balance and short-term nutritional and digestional status to produce an output signal to feeding and autonomic circuits.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery and Cancer, Imperial College London, London, UK.

ABSTRACT

Objective: Glucagon-like peptide 1 (GLP-1) is involved in the central regulation of food intake. It is produced within the brain by preproglucagon (PPG) neurons, which are located primarily within the brain stem. These neurons project widely throughout the brain, including to the appetite centers in the hypothalamus, and are believed to convey signals related to satiety. Previous work demonstrated that they are directly activated by leptin and electrical activity of the afferent vagus. Another satiety hormone, cholecystokinin (CCK), has also been linked to activation of brain stem neurons, suggesting that it might act partially via centrally projecting neurons from the nucleus tractus solitarius (NTS). The aim of this study was to investigate the neuronal circuitry linking CCK to the population of NTS-PPG neurons.

Research design and methods: Transgenic mice expressing yellow fluorescent protein (Venus) under the control of the PPG promoter were used to identify PPG neurons in vitro and to record their electrical and pharmacological profile.

Results: PPG neurons in the NTS were excited by CCK and epinephrine, but not by the melanocortin receptor agonist melanotan II. Both CCK and epinephrine acted to increase glutamatergic transmission to the PPG neurons, and this involved activation of α(1)-adrenergic receptors. Inhibition of adrenergic signaling abolished the excitatory action of CCK.

Conclusions: CCK activates NTS-PPG cells by a circuit involving adrenergic and glutamatergic neurons. NTS-PPG neurons integrate a variety of peripheral signals that indicate both long-term energy balance and short-term nutritional and digestional status to produce an output signal to feeding and autonomic circuits.

Show MeSH
Related in: MedlinePlus