Cholecystokinin Switches the Plasticity of GABA Synapses in the Dorsomedial Hypothalamus via Astrocytic ATP Release

Abstract

Whether synapses in appetite-regulatory brain regions undergo long-term changes in strength in response to satiety peptides is poorly understood. Here we show that following bursts of afferent activity, the neuromodulator and satiety peptide cholecystokinin (CCK) shifts the plasticity of GABA synapses in the dorsomedial nucleus of the hypothalamus of male Sprague Dawley rats from long-term depression to long-term potentiation (LTP). This LTP requires the activation of both type 2 CCK receptors and group 5 metabotropic glutamate receptors, resulting in a rise in astrocytic intracellular calcium and subsequent ATP release. ATP then acts on presynaptic P2X receptors to trigger a prolonged increase in GABA release. Our observations demonstrate a novel form of CCK-mediated plasticity that requires astrocytic ATP release, and could serve as a mechanism for appetite regulation.SIGNIFICANCE STATEMENT Satiety peptides, like cholecystokinin, play an important role in the central regulation of appetite, but their effect on synaptic plasticity is not well understood. The current data provide novel evidence that cholecystokinin shifts the plasticity from long-term depression to long-term potentiation at GABA synapses in the rat dorsomedial nucleus of the hypothalamus. We also demonstrate that this plasticity requires the concerted action of cholecystokinin and glutamate on astrocytes, triggering the release of the gliotransmitter ATP, which subsequently increases GABA release from neighboring inhibitory terminals. This research reveals a novel neuropeptide-induced switch in the direction of synaptic plasticity that requires astrocytes, and could represent a new mechanism by which cholecystokinin regulates appetite.

Keywords: ATP; GABA; appetite; astrocytes; cholecystokinin; dorsomedial hypothalamus.

Figure 1.

CCK shifts the plasticity of GABA synapses from LTD to LTP. A, Schematic of DMH illustrating the approximate position of recording and stimulating electrodes in the compact zone (CZ) of the DMH (f, fornix; 3V, third ventricle). B, Sample traces of averaged IPSCs before (baseline; black trace) and after (post-HFS; gray trace) HFS of afferents (top), and summary data showing a long-lasting reduction in IPSC amplitude (bottom; n = 6; 46.8 ± 13.8% of baseline). Calibration: 50 pA and 10 ms. Amplitude values were assessed during the 5 min before HFS (baseline) and 10–20 min following HFS (post-HFS). Arrow indicates the time of HFS. C, Sample traces of averaged IPSCs before (baseline; black trace) and after HFS (post-HFS; red trace; top) in a representative cell showing a long-lasting potentiation in IPSC amplitude in the presence of CCK (0.1 μm; bottom). D, Summary data showing LTP_CCK following HFS in the presence of CCK (n = 6; 252.2 ± 38.2% of baseline). E, Summary PPR data (left) and CV data (right) showing a decrease following HFS in CCK-treated slices. The horizontal black bar indicates the mean. F, Sample traces of spontaneous IPSCs before (baseline; black trace) and after HFS (post-HFS; red trace) in the continuous presence of CCK and summary data showing an increase in sIPSC frequency, but no change in amplitude following HFS in CCK-treated slices. Calibration: 25 pA and 0.5 s. *p > 0.05.

Figure 2.

CCK2R activation is necessary for LTP_CCK. A, Sample traces of averaged IPSCs before and after HFS (top), and summary data showing that LTP_CCK is completely abolished with the CCK2 receptor antagonist LY-225910 (bottom; 1 μm; n = 8; 100.2 ± 11.36% of baseline). B, Sample traces of averaged IPSCs before and after HFS (top) and summary data showing that LTP_CCK is not prevented with the CCK1R antagonist, lorglumide (bottom; 1 μm; n = 8; 176.4 ± 18.96% of baseline). The horizontal black bar indicates the mean. **p > 0.01. C, Summary data of the percentage change in IPSC amplitude from baseline in control slices and in the presence of CCK alone (0.1 μm; n = 6; 252.2 ± 38.2% of baseline) or with CCK and the following: l-NAME (200 μm; n = 8; 160.7 ± 20.99% of baseline) or GDPβS (1 mm; n = 6; 165.0 ± 14.42% of baseline) in the patch pipette. *p > 0.05, **p > 0.01, ***p > 0.001 compared with baseline for each group.

Figure 3.

Astrocytes are necessary for LTP_CCK. A, Sample traces of averaged IPSCs before and after HFS (top), and summary data showing the depression of GABA synapses in slices incubated in fluorocitric acid in the presence of CCK (bottom; 100 μm; n = 8; 75.4 ± 17.5% of baseline). B, Example image of GCaMP3-expressing astrocytes in the dorsomedial hypothalamus. Scale bar, 10 μm. C, D, Average intensity of field of view before (baseline; C) and after CCK application (0.1 μm; D). Scale bar, 10 μm. E, Example traces of astrocytic intracellular calcium before and after CCK application. Calibration: 0.2 dF/F and 2 min. F, CCK application increased the amplitude of individual calcium events in astrocytes (n = 11). G, CCK application increased the frequency of individual calcium events in astrocytes (n = 11). The horizontal black bar indicates the mean. *p > 0.05.

Figure 4.

LTP_CCK requires mGluR5 activation. A, Sample traces of averaged IPSCs before and after HFS (top), and summary data showing the depression of GABA synapses in the presence of CCK and the group I mGluR antagonist MCPG (bottom; 200 μm; n = 6; 43.3 ± 8.2% of baseline). B, Sample traces of averaged IPSCs before and after HFS (top), and summary data showing the depression of GABA synapses in the presence of CCK and the mGluR5 antagonist MTEP (bottom; 10 μm; n = 6; 54.4 ± 10.9% of baseline). C, Sample traces of averaged IPSCs before and after HFS (top), and summary data showing depression of GABA synapses in the presence of the mGluR5 agonist DHPG (bottom; 10 μm; n = 6; 45.29 ± 12.74% of baseline). The horizontal black bar indicates the mean. *p > 0.05, **p > 0.01, ***p > 0.001.

Figure 5.

ATP-induced activation of presynaptic P2X receptors is required for LTP_CCK. A, Sample traces of averaged IPSCs before and after HFS (top) in a representative cell (bottom) showing a long-lasting depression in IPSC amplitude in the presence of CCK and the P2X receptor antagonist PPADS (30 μm). B, Summary data showing depression in IPSCs following HFS in the presence of CCK and PPADS (n = 6; 51.9 ± 9.1% of baseline). C, Sample traces of averaged IPSCs before and during incubation with the ATP analog ATPγS (100 μm; top) in a representative cell (bottom) showing the potentiation of IPSC amplitude with ATPγS. D, Summary data showing the potentiation of GABA synapses with ATPγS (n = 6; 194.1 ± 27.6% of baseline). The shaded region represents the time and duration of ATPγS application to the slice.

Figure 6.

Schematic representation of the proposed mechanism, as follows: 1, In the presence of CCK, CCK2Rs are activated on astrocytes; 2, HFS triggers the release of glutamate from neighboring neurons, leading to the activation of mGluR5Rs on astrocytes; 3, the synergistic action of CCK2Rs and mGluR5s on astrocytes causes a release of calcium from intracellular stores, resulting in the release of ATP; and 4, ATP binds to P2XRs on GABA terminals, causing a prolonged increase in GABA release onto DMH neurons.