Introduction
Cholecystokinin (CCK), the most abundant and widely distributed neuropeptide in the CNS (Crawley and Corwin, 1994), colocalizes with glutamate (Morino et al., 1994), GABA (Somogyi et al., 1984; Markram et al., 2004), vasoactive intestinal peptide (VIP; Kosaka et al., 1985), dopamine (Hökfelt et al., 1980), and serotonin (Van Der Kooy et al., 1981). Repetitive firing or stimulation of GABAergic neurons, especially at high frequency, induces neuroplasticity of inhibition and long-term potentiation of inhibitory synapses (iLTP) in the neocortex and hippocampus (Komatsu, 1994; Caillard et al., 1999; Nugent et al., 2007). CaMKII-dependent phosphorylation of GABAA receptor (GABAAR)-induced postsynaptic iLTP has been recognized (Petrini et al., 2014; He et al., 2015; Chiu et al., 2018). In addition, cortical interneuron-mediated iLTP is expressed in other forms, including NMDA receptor-dependent spike-timing-dependent plasticity in the auditory cortex (AC; Vickers et al., 2018), brain-derived neurotrophic factor receptor-TrkB signaling-dependent iLTP in the developing AC (Xu et al., 2010), and GABABR-dependent forms of iLTP in the visual cortex (Komatsu, 1996). In the hippocampal network, synapses from parvalbumin (PV)-expressing interneurons (INs; PV-INs) or somatostatin (SOM)-expressing interneurons (SOM-INs) can undergo two unique forms of plasticity relying on T-type voltage-gated Ca2+ channel (VGCC) activation, long-term depression (LTD; PV-iLTD) or SOM-iLTP, respectively (Udakis et al., 2020). These forms of iLTP indicate that different mechanisms are involved depending on brain areas, neuronal connections, and brain activities.
CCK-related inhibitory plasticity has been characterized by the aspects of brain regions, cell types, and synapses. CCK-mediated inhibitory plasticity requires high-frequency stimulation (HFS) as well as CCK2R activation and astrocytic ATP release in the dorsomedial hypothalamus via increased GABA release (Crosby et al., 2015, 2018). CCK increases the frequency of spontaneous ISPCs (sIPSPs) and currents (sIPSCs) in the basolateral amygdala (Chung and Moore, 2007). Recently, Martinez Damonte et al. (2022) reported that HFS-induced somatodendritic-released CCK potentiates GABAergic synapses onto ventral tegmental area dopamine cells. Also, Dudok et al. (2021) found that CCK basket cell activity inversely scales with pyramidal cell ensemble activity during locomotion and rest. However, cortical CCK-IN-induced iLTP is not fully elucidated at the presynaptic or postsynaptic levels. Despite CCK being a marker for cortical GABAergic neurons (Kubota and Kawaguchi, 1997), its functional role in cortical GABA inhibitory synapses is still unknown, as well as which CCK receptor is involved in this process.
CCK1R and CCK2R, two known CCK receptors in the mammalian brain (Moran et al., 1986; Wank et al., 1992; Noble and Roques, 1999), are G-protein-coupled receptors (GPCRs). CCK1R links hippocampal LTP and spatial memory (Harro and Oreland, 1993). CCK2R mediates LTP in excitatory synapses and enables associative memory encoding between two sounds, sound and light, or sound and fear (Li et al., 2014; Chen et al., 2019; Zhang et al., 2020; Feng et al., 2021). Given that CCK mediates excitatory and inhibitory circuit LTP in different brain regions, the following question arises: Does CCK released from GABAergic neurons mediate iLTP in the cortex via known CCK receptors?
We performed in vivo extracellular and in vitro patch-clamp recording experiments to address the above question in the present study. We found that iLTP was not induced in CCK-KO mice but in CCK1R/2R double KO mice, implying that a novel CCK receptor mediates iLTP. Our next objective was to identify this novel CCK receptor. We then screened the homology and similarities of known GPCRs to CCK1R and CCK2R using a bioinformatics algorithm to shortlist candidate receptors, followed by multiple cell-based assays to validate their binding properties with the CCK ligand. We also examined the colocalization of candidate GPCRs and CCK-GABAergic synapses and their distribution in excitatory and inhibitory neurons. Finally, we performed loss-of-function studies in cell and brain slice assays. In this manner, we identified GPR173 as a novel CCK receptor involved in the iLTP of CCK-INs in the cortex.
Results
CCK-INs potentiate inhibition in the AC
We adopted an in vivo recording animal model for the first experiment. Considering activation of GABAergic neurons induces inhibitory outputs to nearby neurons in the neocortex, we assumed prior activation of GABAergic neurons would suppress neuronal responses in the AC to a forthcoming AS.
Conditional CCK-KO (CCK-cKO) mice were generated using the CRISPR/Cas9 system. Vgat-Cre and CCK-cKO mice were crossed to purposefully knock out CCK in GABAergic neurons (CCK−/−-GABA). We injected the same Cre-dependent adeno-associated virus (AAV) vector AAV-EF1a-DIO-ChETA-eYFP in the AC of Vgat-Cre and Vgat-Cre-CCK-cKO mice (Fig. 1A) to infect GABAergic neurons specifically. Most eYFP+ neurons colocalized with GAD67 (Fig. 1B; Vgat-Cre, 76.91 ± 4.03%, n = 12 sections, N = 5 mice; Vgat-Cre-CCK-cKO, 76.17 ± 1.92%, n = 11 sections, N = 5 mice, one-way ANOVA with Tukey’s post hoc test, N.S.).
HFLS of GABAergic CCK neurons enhances their inhibition in the long term. A, Schematic illustration of Cre-dependent AAV vector (DIO-ChETA-eYFP) injection into the AC of Vgat-Cre and Vgat-Cre-CCK-cKO mice to specifically infect GABAergic neurons. B, Immunohistochemical labeling images of eYFP, GAD67, and DAPI in the AC of DIO-ChETA-eYFP-injected Vgat-Cre and Vgat-Cre-CCK-cKO mice. Scale bars: 100 and 20 µm, respectively. The bar chart depicts the percentage of colabeled neurons positive for eYFP and GAD67 in virus-injected Vgat-Cre (n = 12 sections, N = 5 mice) and Vgat-Cre-CCK-cKO mice (n = 11 sections, N = 5 mice; one-way ANOVA with Tukey’s post hoc test; N.S., not significant). C, The setup for in vivo recordings with laser stimulation in the AC (natural AS, a noise burst; SOM, PV, and PC). D, Left to right, Unit response in raster displays and PSTHs to the AS with the laser off (left) or on (right), and group data for mean frequency of the AS-triggered firing rate with the laser off or on (DIO-ChETA-eYFP-injected mice, n = 17 units, N = 11 mice; two-way ANOVA with Tukey’s post hoc test, ***p < 0.001). E, Multiunit recording in Vgat-Cre mice. Left, Unit response (middle) and mean firing rate (right) of responses to the combined stimulus of the laser and AS before, during, and after HFLS in Vgat-Cre mice. F, Multiunit recording in Vgat-Cre-CCK-cKO mice. Left to right, Unit response and mean firing rate (right) for responses to the combined stimulus of the laser and AS before, during, and after HFLS in Vgat-Cre-CCK-cKO mice. G, Group data for E, F [Vgat-Cre (red), n = 16 units, N = 11 mice; Vgat-Cre-CCK-cKO (blue), n = 14 units, N = 11 mice; two-way ANOVA with Tukey’s post hoc test; ***p < 0.001, N.S.; Vgat-Cre vs Vgat-Cre-CCK-cKO, two-way ANOVA with Tukey’s post hoc test, ##p < 0.01]. H, Unit response and mean spontaneous firing rate before and after HFLS in DIO-ChETA-eYFP–injected Vgat-Cre (n = 16 units, N = 11 mice) and Vgat-Cre-CCK-cKO mice (n = 14 units, N = 11 mice; two-way ANOVA with Tukey’s post hoc test, N.S.). I, LFP response to the AS (with a preceding laser pulse). Representative traces before (dashed lines) and after (solid lines) HFLSs were recorded in Vgat-Cre (red) and Vgat-Cre-CCK-cKO (blue) mice. J, Time course of the normalized amplitude of LFP responses to the AS (with a preceding laser pulse) before and after HFLS [Vgat-Cre-CCK-cKO (blue), n = 16 recording sites, N = 11 mice; Vgat-Cre (red), n = 14 recording sites, N = 11 mice, two-way RM ANOVA with Tukey’s post hoc test, ****p < 0.0001, ####p < 0.0001, N.S., not significant]. Data indicate mean ± SEM.
In the in vivo setup for anesthetized mice (Fig. 1C), normalized sound intensity, and laser power were selected after measuring the input-output curve of each mouse for stimulus intensities. Also, the kinetics of optogenetic opsin were examined by applying laser pulse trains of different frequencies (1–120 Hz) in both groups to ensure normal firing of GABAergic neurons after knocking out CCK.
Laser stimulation significantly suppressed evoked response to the forthcoming AS in ChETA-eYFP-injected Vgat-Cre mice (Fig. 1D, left), response to the AS in raster displays (90 trials over 15 min), and poststimulus histograms (PSTHs; bin width, 10 ms; middle, response to the AS preceded by laser stimulation; right, mean auditory response calculated in a 100 ms observation window starting 20 ms after the AS in ChETA-eYFP-injected mice, which was reduced from 152.23 ± 29.22 to 104.07 ± 20.75 Hz (n = 17 units; two-way ANOVA with Tukey’s post hoc test, ***p < 0.001). Activation of local GABAergic neurons in the AC inhibited responses to the forthcoming natural AS in the AC.
Next, we examined whether HFLS of GABAergic neurons could potentiate their inhibition of neuronal responses to the forthcoming AS in the AC and whether CCK-INs are involved in potentiating their inhibitory outputs toward their target neurons.
Laser-activated GABAergic neurons showed inhibited neuronal responses to the forthcoming AS in Vgat-Cre mice (Fig. 1E–F). We adopted the HFLS protocol (five-pulse bursts at 40 Hz, 10 s interburst interval, 200 pulses in total). HFLS of GABAergic neurons potentiated the inhibitory effect of laser-activated GABAergic neurons on neuronal responses to the forthcoming AS in Vgat-Cre mice (Fig. 1E, from left to right, before and after HFLS and group data for firing rate in raster displays and PSTHs). We found no potentiation of inhibition in Vgat-Cre-CCK-cKO mice, which showed similar laser-evoked responses as Vgat-Cre mice before, during, and after HFLS [Fig. 1F, from left to right, rasters before, during, and after HFLS in Vgat-Cre-CCK-cKO mice; Fig. 1G, group firing rates before and after HFLS, Vgat-Cre mice (red), 113.08 ± 36.92 and 63.86 ± 17.82 Hz, n = 16 units, N = 11 mice, two-way ANOVA with Tukey’s post hoc test, ***p < 0.001; Vgat-Cre-CCK-cKO (blue), 116.63 ± 17.79 and 120.97 ± 16.98 Hz, n = 14 units, N = 11 mice; two-way ANOVA with Tukey’s post hoc test; N.S.; Vgat-Cre, 63.86 ± 17.82 Hz vs Vgat-Cre-CCK-cKO, 120.97 ± 16.98 Hz, two-way ANOVA with Tukey’s post hoc test, ##p < 0.01]. The spontaneous firing rates of the two groups remained the same before and after HFLS (Fig. 1H). These results suggest enhanced inhibition occurred when neuronal responses exceeded a certain excitatory threshold.
The enhanced inhibition was also reflected in LFP responses. Example traces in Figure 1I and group data in Figure 1J show that HFLS induced a distinct reduction in the amplitude of LFPs to the AS in Vgat-Cre mice but not in Vgat-Cre-CCK-cKO mice (Fig. 1J; Vgat-Cre, 52.75 ± 6.84% vs Vgat-Cre-CCK-cKO, 103.13 ± 2.34%, two-way RM ANOVA with Tukey’s post hoc test, ####p < 0.0001).
In summary, HFLS of local GABAergic neurons potentiated the inhibitory effect of GABAergic neurons on neuronal responses to the forthcoming AS in Vgat-Cre mice but not in Vgat-Cre-CCK-cKO mice. The potentiated inhibition was long lasting and persisted across the observation period of 60 min (Fig. 1J).
HFLS of CCK-INs, but not PV-INs, potentiates sIPSCs in glutamatergic neurons
Earlier studies on cortical inhibitory neuron-related plasticity and behaviors extensively focus on discriminating the distinct roles of PV-IN, SOM-IN, and VIP-IN-induced inhibitory control (Kvitsiani et al., 2013; Pfeffer et al., 2013; Pi et al., 2013; Chen et al., 2017; Yu et al., 2019; Jang et al., 2020) because they account for the majority of neocortical GABAergic neurons (Gonchar, 2008; Whissell et al., 2015) and are easily characterized by selective genetic and histologic labeling. In contrast, cortical CCK-INs constitute a major subclass of GABAergic interneurons but receive less attention because of difficulties isolating CCK-INs.
The above in vivo experiments show that HFLS of GABAergic neurons in Vgat-Cre mice but not in Vgat-Cre-CCK-cKO mice potentiated the inhibition of neuronal activity in the AC. In the next experiment, we performed a patch-clamp recording of pyramidal neurons to examine whether the potentiated inhibition of GABAergic inputs was CCK-IN specific. We targeted CCK-INs and considered PV-INs as a control as PV-INs represent the largest proportion of GABAergic neurons in the cortex and hippocampus and serve to gate perisomatic inhibition (Gonchar, 2008; Whissell et al., 2015; Dudok et al., 2021). Dudok et al. (2021) specifically labeled hippocampal CA1 CCK basket cells Sncg (58 ± 17%), but no other inhibitory subtypes using the Sncg-Flp mouse line (Dudok et al., 2021). To unbiasedly label cortical CCK-INs and PV-INs, we combined a Cre recombinase-based genetic approach with the inhibitory neuron-specific intergenic regulatory sequence Dlx5/6 (Whissell et al., 2015), which provides selective genetic access and sufficient labeling in available PV-Cre and CCK-Cre transgenic mouse lines (Taniguchi et al., 2011).
To virally target PV-INs or CCK-INs to a comparable extent, we injected the Cre-dependent AAV virus into the AC of PV-Cre or CCK-Cre mice. We designed a Channelrhodopsin-2 (ChR2) and mCherry-expressing AAV under the control of the inhibitory neuron-specific Distal-less homeobox (mDlx) gene enhancer (rAAV9-mDlx-DIO-ChR2-mCherry-WPRE-pA, hereon referred to as mDlx-DIO-ChR2-mCherry; Fig. 2A). mDlx enhancer expression is specific to and robust in cortical interneurons across vertebrate species (Dimidschstein et al., 2016). Our virus labeled mostly CCK-INs or PV-INs in CCK-Cre or PV-Cre mice, respectively (Fig. 2B; 96.3% of mCherry-labeled neurons were GAD67+, 95.4 ± 1.6% were CCK+, and 96.7 ± 1.2% were ChR2+ in CCK-Cre mice; 97.5 ± 1.0% of mCherry-labeled neurons were PV+ and 98.2 ± 1.0% were ChR2+ in PV-Cre mice).
HFLS of GABAergic CCK synapses potentiates IPSCs in postsynaptic glutamatergic neurons. A, Injection of rAAV9-mDlx-DIO-ChR2-mCherry into CCK-Cre and PV-Cre mice. B, Images for mCherry colabeled with the indicated markers (GAD67, ChR2, PV, and CCK) in the AC of CCK-Cre or PV-Cre mice. Scale bars: 20 µm. The bar chart depicts the percentage of mCherry+ neurons colabeled with GAD67, ChR2, and CCK in CCK-Cre mice or colabeled with PV and ChR2 in PV-Cre mice. C, Schematic drawings show in vitro patch-clamp recording of target PC neurons while delivering a laser pulse to activate infected CCK neurons (top) and PV neurons (bottom). The confocal image depicts the morphology of a representative pyramidal neuron infused with Alexa Flour 488 after patch-clamp recording in CCK-Cre mice. Scale bar, 15 µm. Neuronal firing responses to step currents show the recruited PC with regular spiking. D, Traces of IPSC responses to the laser pulse at 0.1 Hz in brain slices from CCK-Cre and PV-Cre mice before and after HFLS. E, Sampled neuronal responses to the HFLS in PV-Cre and CCK-Cre brain slices. F, Time course of normalized IPSCs before (for 5 min) and after (for 25 min) HFLS in PV-Cre (black, n = 9 neurons; one-way ANOVA with Tukey’s post hoc test; N.S.) and CCK-Cre (red, n = 9 neurons; one-way ANOVA with Tukey’s post hoc test, ****p < 0.0001; two-way RM ANOVA with Tukey’s post hoc test, ####p < 0.0001) mice. G, Schematic drawings show patch-clamp recording targeted at pyramidal neurons while delivering a laser pulse to activate infected neurons and perfusing CCK8s or aCSF. Neuronal responses to step currents were used to confirm that recorded neurons were excitatory with regular spike trains as in C. H, IPSCs in response to the laser pulse in brain slices from CCK-Cre mice perfused with CCK8s or aCSF. I, Time course of normalized IPSCs before (for 5 min) and after (for 25 min) LFLS in brain slices from CCK-Cre mice perfused with aCSF (blue, n = 8 neurons; one-way ANOVA with Tukey’s post hoc test, N.S., not significant) or CCK8s (red, n = 8 neurons; one-way ANOVA with Tukey’s post hoc test, ****p < 0.0001; two-way RM ANOVA, post hoc with Tukey HSD, ####p < 0.0001). Data are expressed as mean ± SEM.
Brain slices from CCK-Cre and PV-Cre mice were used for in vitro patch-clamp recording (Fig. 2C). First, we selectively patched pyramidal neurons. We chose pyramidal-shaped cells that emitted regular spike trains in response to current-step injection, as in previous studies (Connors and Gutnick, 1990; Stiefel et al., 2013). In Figure 2C, the morphology of the pyramidal cell was visualized by Biotin 488. The physiological properties were reflected by the responses to injected step currents, after which IPSCs (with a short latency of 4.53 ± 0.28 ms, n = 10 cells) evoked by laser stimulation (0.15 Hz, 5 min as baseline) were directly recorded. IPSCs in CCK-Cre mice were potentiated robustly after HFLS, whereas no potentiation of IPSCs occurred in PV-Cre mice (Fig. 2D; single, and averaged traces of IPSCs in response to laser stimulation at 0.15 Hz before and after HFLS during 0–5 min, 5–15 min, and 15–25 min recording periods; Fig. 2E, representative neuronal responses to HFLS). The time course of normalized IPSCs before (for 5 min) and after (for 25 min) HFLS showed no significant potentiation of IPSCs in PV-Cre mice (Fig. 2F; baseline vs 20–25 min after HFLS, 100.00 ± 2.43% vs 103.67 ± 7.00%, n = 7 neurons, two-way ANOVA with Tukey’s post hoc test, p = 0.26, N.S.), whereas HFLS of CCK-INs induced significant potentiation of IPSCs in CCK-Cre mice (Fig. 2F; baseline vs 20–25 min after HFLS, 101.56 ± 5.73% vs 183.70 ± 6.25%, n = 9 neurons, one-way ANOVA with Tukey’s post hoc test, ****p < 0.0001).
In summary, HFLS of CCK-INs induced significant potentiation of IPSCs during the recording period, or iLTP (HFLS of CCK-INs vs HFLS of PV-INs, 183.70 ± 6.25% vs 103.67 ± 7.00%, two-way RM ANOVA with Tukey’s post hoc test, ####p < 0.0001).
Application of CCK8s and lLFLS to CCK-INs induces iLTP in glutamatergic neurons
Next, we hypothesized that HFLS of CCK-INs triggers the corelease of GABA and CCK that induces the iLTP of their target neurons. At the same time, LFLS with the same number of pulses causes no CCK release and, thus, no iLTP. In this case, the direct application of CCK followed by LFLS could generate iLTP in CCK-INs.
To test this hypothesis, we adopted the same in vitro patch-clamp recording method in pyramidal neurons in the AC with CCK-INs selectively transfected with AAV virus (mDlx-DIO-ChR2-mCherry; Fig. 2G). We applied CCK8s or aCSF followed by LFLS (Fig. 2H). We observed large iLTP in the CCK8s + 1 Hz group and no iLTP in the aCSF + 1 Hz group (Fig. 2H, single and averaged traces of IPSCs; Fig. 2I, normalized IPSC amplitudes; baseline vs 20–25 min after CCK8s + 1 Hz, 100.00 ± 2.53% vs 161.84 ± 4.38%, n = 8 neurons; baseline vs 20–25 min after aCSF + 1 Hz, 100.10 ± 1.96% vs 92.05 ± 6.58%, n = 7 neurons, two-way ANOVA with Tukey’s post hoc test, ****p < 0.0001; N.S.).
Therefore, iLTP in CCK-INs was induced by the application of CCK8s followed by LFLS but not in the aCSF control group (20–25 min after aCSF + 1 Hz vs 20–25 min after CCK8s + LFLS, two-way RM ANOVA with Tukey’s post hoc test, ####p < 0.0001).
HFLS induces iLTP in CCK1R/2R-KO mice
Next, we aimed to determine which CCK receptor mediates iLTP in CCK-INs. There are two known CCK receptors in the brain, CCK1R and CCK2R. CCK2R is widely distributed throughout the CNS, whereas CCK1R is only found in certain regions, such as the hypothalamus and hippocampus (Honda et al., 1993). We reported that CCK2R mediates the role of entorhinal-cortical CCKs in cortical LTP and the encoding of associative memory (Li et al., 2014; Chen et al., 2019). Thus, we expected that CCK-induced iLTP is mediated by CCK receptors.
We used CCK1R/2R-KO and CCK-KO mice to examine which CCK receptor mediates the contribution of CCK signaling to the iLTP of CCK-INs in brain slice experiments. We performed genotyping confirmation of CCK1R/2R-KO and CCK-KO mouse lines before virus injection and in vitro recording.
We combined two viruses (Fig. 3A; mDlx-DIO-ChR2-mCherry and mDlx-Cre-helper rAAV9) that both contain mDlx and are expressed only in interneurons. The viruses were injected into the AC of CCK1R/2R-KO and CCK-KO mice.
Known CCK receptors do not mediate CCK signaling in CCK-INs. A, Schematic drawings show rAAV9-mDlx-DIO-ChR2-mCherry-WPRE-pA (mDlx-DIO-ChR2-mCherry) and AAV9-mDlx-Cre-WPRE-pA (mDlx-Cre) injected into CCK1R/2R-KO or CCK-KO mice. B, AAV9-infected mCherry neurons colabeled with GAD67 (left, CCK1R/2R-KO mouse; right, CCK-KO mouse). Scale bars: 50 and 10 µm, respectively. Right, Bar chart shows the percentage of mCherry neurons colabeled with GAD67 in CCK1R/2R-KO and CCK-KO mice. C, Schematic drawings show in vitro patch-clamp recordings from pyramidal neurons while delivering a laser pulse to activate infected GABAergic neurons in CCK-KO and CCK1R/2R-KO mice. Neuronal responses to a step current confirmed that the recorded neurons were excitatory. D, Traces of IPSCs in response to the laser pulse in CCK1R/2R-KO (top) and CCK-KO (bottom) mice before and after HFLS of infected GABAergic neurons. E, Sampled neuronal responses to HFLS in CCK1R/2R-KO and CCK-KO mice. F, Time course of normalized IPSCs before and after (for 25 min) HFLS in CCK-KO (black, n = 9 neurons; one-way ANOVA with Tukey’s post hoc test, N.S., not significant) and CCK1R/2R-KO mice (green, n = 9 neurons; one-way ANOVA with Tukey’s post hoc test, ****p < 0.0001; two-way RM ANOVA with Tukey’s post hoc test, ####p < 0.0001). G, Schematic illustration of the setup for in vivo recordings with laser stimulation in the AC and a natural AS (a noise burst; SOM, PV neuron, PC) in CCK1R/2R-KO (left) and CCK-KO (right) mice. Bottom, Example waveforms from the two groups. H, Normalized LFP response amplitudes to the AS with a preceding laser pulse before and after HFLS in CCK1R/2R-KO (green, n = 7 recording sites, N = 5 mice) and CCK-KO (black, n = 8 recording sites, N = 6 mice; two-way RM ANOVA with Tukey’s post hoc test, ****p < 0.0001) mice. Data are expressed as mean ± SEM.
Immunohistochemical experiments examined whether the combined viruses were expressed in the target GABAergic neurons effectively and precisely. Confocal images showed that almost all infected neurons were GABAergic neurons in both mouse lines (Fig. 3B; one-way ANOVA with Tukey’s post hoc test, N.S.).
In the following experiments, pyramidal neurons were selected for recording using in vitro patch clamping (Fig. 3C; preparation for recording and responses to current injection). HFLS of GABAergic neurons induced iLTP in recorded pyramidal neurons in CCK1R/2R-KO mice but not in CCK-KO mice (Fig. 3D, single and averaged traces; Fig. 3E, IPSCs in response to HFLS, 40 Hz, 10 pulses for both CCK1R/2R-KO and CCK-KO mice; Fig. 3F, CCK1R/2R-KO mice, baseline vs 20–25 min after HFLS, 100.36 ± 2.91% vs 150.34 ± 5.32%, n = 9 neurons, two-way ANOVA with Tukey’s post hoc test, ****p < 0.0001; CCK-KO mice, baseline vs 20–25 min after HFLS, 99.93 ± 2.22% vs 97.71 ± 2.98%, n = 9 neurons, two-way ANOVA with Tukey’s post hoc test, p = 0.82, N.S.).
We then decided to confirm these iLTPs using the same protocol for in vivo recording in CCK1R/2R-KO mice (Fig. 3G). We examined whether laser activation of GABAergic neurons suppresses neuronal responses in the AC to a forthcoming AS. As expected, HFLS of GABAergic neurons did not reduce the amplitude of LFPs in response to the AS in CCK-KO mice. To our surprise, HFLS of GABAergic neurons induced a distinct decrease in the amplitude of LFPs in response to the AS in CCK1R/2R-KO mice (Fig. 3H; CCK1R/2R-KO, 63.56 ± 5.09% vs CCK-KO, 100.17 ± 4.20%, two-way RM ANOVA with Tukey’s post hoc test, ####p < 0.0001), indicating that a novel CCK receptor other than CCK1R or CCK2R is likely involved in mediating the signaling of CCK from CCK-INs.
These results indicate that CCK-enabled iLTP is likely mediated by a novel receptor other than CCK1R or CCK2R.
Bioinformatics and binding assay search for potential CCK receptors
Currently, five categories of methods are mainly being used to characterize the binding of GPCRs and ligands based on their detection mechanisms, namely, direct binding assay (Stoddart et al., 2015; Flanagan, 2016; Rice et al., 2019), G-protein-based signal detection (Seifert et al., 1999; Inoue et al., 2012; Ma et al., 2017), arrestin-dependent signal detection (Bassoni et al., 2012), biophysical methods (Navratilova et al., 2011; Schroder et al., 2011; Rajarathnam and Rosgen, 2014), and bioinformatics prediction (Kharkar et al., 2014; Foster et al., 2019).
The downstream signals of many GPCRs are unclear, especially for >100 orphan GPCRs (Laschet et al., 2018). Bioinformatics prediction is the most efficient method of finding GPCR-ligand pairs and hence was adopted in our study to search for novel CCK receptors in the brain. Combining cell-based assays with bioinformatics prediction has enabled the identification of many GPCR-ligand pairs, such as bombesin receptor 3 (BB3)-neuromedin B, BB3-gastrin-releasing peptide, GPR15-GPR15L47-57, GPR68-Osteocrin33-55, and GABABR1a-sAPPa (Foster et al., 2019; Rice et al., 2019). In addition, loss-of-function and gain-of-function studies coupled with histology verification are very important for exploring the physiological functions of novel GPCRs.
Recently, highly accurate protein 2D alignment or 3D structure prediction has facilitated the development of new drugs targeting GPCRs (Insel et al., 2019; Yang et al., 2021). The online alignment of the 2D protein sequence (Sievers et al., 2011) and prediction of the 3D structure by AlphaFold2 or ColabFold systems (Senior et al., 2020; Du et al., 2021; Tunyasuvunakool et al., 2021; Mirdita et al., 2022; Varadi et al., 2022) enables us to map CCK receptors rapidly and comprehensively. We speculated that a new CCK receptor mediates CCK8s-induced iLTP in the mouse AC, which is probably a GPCR similar to CCK1R/2R. The 2D sequences of CCK receptors should be evolutionarily homologous. We predicted the secondary structure of amino acid sequences of selected brain GPCRs using the free online software NetSurfP version 1.1 (Petersen et al., 2009). The binding sites of all GPCR secondary structures (extramembrane) were selected based on the CCK binding domains of CCK1R and CCK2R, and the GPCRs were compared with the two CCK receptors based on hamming distance. We listed the GPCRs with the highest scores (Fig. 4A) as probable candidates for CCK receptors.
Bioinformatics and cell surface binding assay screening for a novel CCK receptor. A, Bar chart showing the highest similarity scores between GPCRs and known CCK receptors (CCK1R and CCK2R) considering the secondary structure in amino acid sequences, including six GPCRs for 5-hydroxytryptamine (serotonin; HTR2C, HTR1F, HTR2B, HTR1E, HTR1B, and HTR1D), an orphan receptor (GPR173), neuropeptide Y receptor Y5 (NPY5R), and adrenergic receptor beta 1 (ADRB1). B–D, Schematic of cell surface binding assay for Flag-tagged GPCRs with negative control (HA-(ε-Ahx)2) or HA-(ε-Ahx)2-CCK variants (HA-CCK4, HA-CCK8ns, and HA-CCK8s). Confocal images (B, top; scale bar, 10 µm) and quantification of fluorescence intensity ratio (Fanti-HA/Fanti-Flag; B, D, bottom) in the cell surface binding assay for Flag-CCK2R, Flag-GPR173, Flag-HTR1B, and five other Flag-GPCRs as shown in C. The number of total cells from three independent experiments is defined as n; n = 33–40, one-way ANOVA with Tukey’s post hoc test or unpaired Student’s t test, **p < 0.01, ****p < 0.0001, N.S., not significant. Data are expressed as mean ± SEM.
To further evaluate which of these GPCR candidates is a novel CCK receptor, we established a cell surface binding assay according to previous reports (De Wit et al., 2013; Rice et al., 2019; Fig. 4B). In this system, the CDS of candidate GPCRs, as well as CCK2R with an N-terminal Flag tag, were cloned into pCDNA3.1(+) to transfect HEK293T cells (Fig. 4B–D). We also synthesized a series of HA-(ε-Ahx)2-CCK variant probes, including CCK4, CCK8s, nonsulfated CCK8 (CCK8ns), and control HA-(ε-Ahx)2, which have been detected in the body with different specificity for CCK1R/2R.
As CCK8s is the main form of CCK in the brain (Dockray et al., 1978; Agersnap et al., 2016), we applied a cell surface binding assay with HA-tagged CCK8s for the first-round screening of GPCR candidates with high scores, including Flag-tagged GPR173, HTR2C, HTR1F, HTR2B, HTR1E, HTR1B, and NPY5R (Fig. 4B,C). We demonstrated that HA-tagged CCK4, HA-tagged CCK8ns, and HA-tagged CCK8s colocalized with Flag-tagged CCK2R, with a significantly larger ratio of CCK ligand HA-tag signal to CCK2R Flag signal (Fanti-HA/Fanti-Flag) than that of the HA-(ε-Ahx)2 control group, suggesting that our cell surface binding assay was applicable for characterizing the binding of CCK ligands and receptors.
CCK-2R and GPR173 bound to CCK8s, whereas other GPCR candidates did not (Fig. 4B–D). Thus, detailed ligand specificity analysis was performed using HA-tagged CCK4/CCK8ns/CCK8s probes, in which HTR1B-expressing cells served as negative/background control. Flag-tagged GPR173 could only bind to HA-tagged CCK8 but not HA-tagged CCK4 or CCK8ns (Fig. 4B), indicating that CCK8s is a specific ligand of GPR173. No binding of HA-tagged CCK4/CCK8ns/CCK8s with Flag-tagged HTR1B was detected. To further verify the binding between GPR173 and CCK8s, we used a competitive cell surface binding procedure, in which Flag-tagged GPR173-expressing HEK293T cells were incubated with a CCK8s and HA-tagged CCK8s (1:1) mixture. We found that CCK8s significantly blocked the HA-tag signals from binding HA-tagged CCK8s (Fig. 4B–D). Together, we conclude that GPR173 is a novel CCK receptor, and CCK8s is a ligand of GPR173.
GPR173 is located at CCK-GABAergic synapses in the AC
The above evidence confirmed that GPR173 is a novel CCK receptor. Here, we performed histochemical analysis and used another bioinformatics prediction algorithm to verify whether GPR173 or another novel CCK receptor mediates HFLS-induced iLTP.
In the bioinformatics prediction algorithm, we used the Clustal Omega program (Sievers et al., 2011) to extract homologous information for 79 orphan receptors from class A GPCRs aligned with the two known CCK receptors. The percentage identity correlation score defines the quantitative correlation estimation between two sets of sequences (Raghava and Barton, 2006). According to the percentage identity correlation score among 79 orphan receptors and CCK1R/2R, we selected top-scored orphan receptors normalized by CCK1R and CCK2R (Fig. 5A). Interestingly, GPR83, which was recently reported as a novel CCK receptor (Mack et al., 2022), was also among the five top-scored candidates (Fig. 5A).
Bioinformatics and histology screening for a novel CCK receptor. A, Percentage identity correlation matrix of orphan receptors with CCK1R/2R. B, Left, Illustration of mDlx-DIO-ChR2-mCherry injected into the AC of CCK-Cre mice. Middle, Immunochemistry staining images of candidate receptors in CCK-GABAergic synapses. Scale bars: 5 µm. Expanded images are from the white rectangles (top right). The white arrows indicate the colocalization of GPR173 with CCK-GABAergic synapses. Scale bars: 1 µm. C, Bar chart shows numbers of overlay dots [bottom right, mCherry+, Syp (synaptophysin)+, and GPCR+, per mm2; n = 3 independent experiments; one-way ANOVA with Tukey’s post hoc test, ****p < 0.0001]. D, Confocal images of Flag-GPR173-transfected HEK293T cells colabeled with anti-GPR173, anti-Flag, and DAPI. Scale bar, 10 µm. Quantitative analysis of colocalization of Flag and GPR173 across the dotted line (labeled in confocal images). The bar chart depicts the percentage of colabeled cells positive for Flag and GPR173 from Flag-GPR173–transfected HEK293T cells stained with anti-GPR173, anti-Flag, and DAPI (n = 21 cells). E, Super-resolution image of colocalized GPR173 with CCK-GABAergic synapses, GABAAR with CCK-GABAergic synapses, and GABAAR and GPR173 (left). Scale bar, 1 µm. The values for PCC and MOC indicate the colocalization possibility of mCherry with GPR173, mCherry with GABAAR, and GABAAR with GPR173. F, Immunochemistry images of costaining of GPR173 with CaMKII (top) or GAD67 (bottom) in C57BL/6J mice. Scale bar, 50 µm. Group data from colocalization analysis of GPR173 with CaMKII (n = 11 sections, N = 3 mice) or GAD 67 (n = 10 sections, N = 3 mice). G, Immunochemistry images for colocalized GPR173 with CCK-GABAergic synapses contacting pyramidal cells and box plot showing numbers of colocalized GPR173 and CCK-GABAergic contacts per pyramidal cell (n = 49 neurons, 5.69 ± 0.38). Scale bar, 5 µm. Data are expressed as mean ± SEM.
The next important question was whether GPR173, GPR83, or other new CCK receptors mediate CCK-dependent iLTP. If so, the new CCK receptors should localize postsynaptically at CCK-GABAergic synapses. We injected mDlx-DIO-ChR2-mCherry AAV in the CCK-Cre mouse AC to label CCK-INs (Fig. 5B, left). We performed triple-labeling of mCherry, synaptophysin, and the GPCR candidates with the highest correlation scores. GPR173 had the highest colocalization density (Fig. 5C; GPR173 vs GPR85, the highest among other GPCRs, 948.90 ± 35.36 vs 53.46 ± 17.68 counts/mm2, one-way ANOVA with Tukey’s post hoc test, ***p < 0.0001) with CCK-GABAergic synapses when compared with 15 other GPCRs—GPR83, CCK1R, CCK2R, GPR85, GPR15, BRS3, GPR17, GPR135, GPR132, TAAR9, GPR19, GPR34, GPR45, TAAR5, and GPR20—suggesting that GPR173 is the most likely CCK receptor mediating HFLS-induced iLTP. To verify the specificity of the GPR173 antibody, pCDNA3.1(+)-Flag-GPR173 was used to transfect HEK293T cells and perform immunofluorescence staining with anti-GPR173 (conjugated with goat anti-rabbit Alexa Fluor 647) and anti-Flag (conjugated with goat anti-mouse Alexa Fluor 568) antibodies (Fig. 5D). Most of the GPR173+ cells were also Flag+ cells (overlay cells, Flag+ cells = 95.23%). We applied a normal-intensity excitation laser and exposure time to both channels to avoid spectral bleed-through. Some Flag+ cells were not GPR173+ in parallel control experiments.
We also checked whether GPR173 colocalizes with GABAAR in mDlx-DIO-ChR2-mCherry-injected CCK-Cre mice using super-resolution imaging with a STORM confocal microscope (Fig. 5E). We found that both GPR173 and GABAAR appeared within mCherry-labeled CCK-GABAergic synapses, and GPR173 and GABAAR also nearly colocalized (Fig. 5E). To determine the co-occurrence and correlation coefficients of GPR173, GABAAR, and mCherry-labeled CCK-GABAergic synapses, we further analyzed data from super-resolution images using PCC and MOC, which are common metrics for measuring the predictability of colocalization (Aaron et al., 2018; Fig. 5E). The numerical values of PCC and MOC were >0.78 across six clusters, suggesting that GPR173 is located in GABAergic synapses and near GABAARs.
Moreover, as previous in vivo and in vitro recording studies described, HFLS-induced iLTP occurs in excitatory pyramidal neurons. We performed costaining of GPR173 with CaMKII (a marker of pyramidal neurons) or GAD67 (a marker of inhibitory neurons; Fig. 5F). More than 90% of GPR173+ cells were CaMKII+ neurons (91.27 ± 1.48% of GPR173+ cells were CaMKII+ neurons, n = 11 sections from 3 C57BL/6J wild-type mice). However, only a small number of GPR173+ cells were costained with GAD67 in the AC (Fig. 5F; 9.82 ± 1.42% were GAD67+, n = 10 sections from 3 C57BL/6J wild-type mice).
To further confirm that CCK-GABAergic synapses directly contact excitatory pyramidal neurons, we counted numbers of colocalized GPR173 and CCK-INs contacts on CaMKII+ neurons (Fig. 5G; n = 49 neurons, 5.69 ± 0.38) in cortical slices from CCK-Cre mice (as described in Fig. 5B). All these data suggest that GPR173 is located at CCK-GABAergic synapses and might be involved in CCK-dependent HFLS-induced iLTP. In the present study, we cannot exclude the possibility that CCK-IN–induced iLTP is restricted to the CCK postsynaptic area without affecting neighboring PV synapses. It would be worth investigating whether this iLTP is homosynaptic or heterosynaptic.
CCK8s triggers intracellular Ca2+ mobilization through GPR173
CCK1R and CCK2R could couple to Gq protein, through which CCK1R/2Rs trigger intracellular Ca2+ release from the endoplasmic reticulum after their activation (Dunlop et al., 1996; Dufresne et al., 2006). CCK8s, as a potent but nonselective agonist of both CCK1R and CCK2R (Berna et al., 2007), mainly works via the Gq-PLC-IP3-Ca2+ pathway (Dunlop et al., 1996; Wu et al., 1997). We hypothesized that the novel CCK receptor should also trigger intracellular Ca2+ mobilization through this pathway on binding to CCK8s. Thus, we built a cell-based Ca2+ imaging assay by constructing stable monoclonal GPCR transgenic CHO cell lines for CHO-GPR173, CHO-CCK1R, and CHO-CCK2R.
In this assay, we used a microplate reader to monitor Ca2+ mobilization (Fig. 6A). As expected, CCK8s could induce a dose-dependent response in CHO-CCK1R/2R cells (Fig. 6B; EC50 CCK1R = 1.72 ± 1.11 nm, EC50 CCK2R = 4.64 ± 2.37 nm). Then, we used the same methods for GPR173 cells and found that CCK8s triggered intracellular Ca2+ mobilization with a high potency of EC50 GPR173 = 3.23 ± 1.94 nm (Fig. 6C), which was almost the same as that for CCK1R/2R.
CCK8s could trigger intracellular Ca2+ mobilization through GPR173 in CHO-GPR173 cells. A, Schematic illustration of Ca2+ imaging assay in CHO-GPCR cell lines. B, Dose-dependent Ca2+ responses of CHO-CCK1R (left; EC50 = 1.72 ± 1.11 nm) and CHO-CCK2R (right; EC50 = 4.64 ± 2.37 nm) cells provoked by CCK8s. C, EC50 curve of CCK8s for the CHO-GPR173 cell line (EC50 = 3.23 ± 1.94 nm). D, Dose-dependent Ca2+ response of CHO-GPCR cells provoked by the CCK2R agonist CCK4. E, Dose-dependent Ca2+ response of CHO-GPCR cells provoked by the CCK1R-specific agonist A-71 623. F, Schematic illustration of knockdown of GPR173 expression in CHO-GPR173 cells using an shRNA of Gpr173. G, 200 nm CCK8s provoked the Ca2+ response in the scramble or shRNA-infected CHO-GPR173 cells and Gpr173 mRNA expression level in the scramble or shRNA-infected CHO-GPR173 cells. H, Ca2+ signal responses of CHO-GPR173 cells treated with 10 μm PNX or 200 nm CCK8s. Relative fluorescence unit (RFU) is defined as the ratio of RFU before and 30 s after adding CCK8s/HHBS (unpaired Student’s t test, *p < 0.05,****p < 0.0001, N.S., not significant). I, Illustration of the β-arrestin1/2 recruitment assay design. J, Dose-dependent response of CCK8s-induced β-arrestin recruitment to CCK1Rs determined by the β-arrestin2 recruitment and β-arrestin1/2 recruitment assays. The two control groups were without GPCR plasmid transfection in HTLA cells. K, L, Dose-dependent response of CCK8s-induced β-arrestin recruitment to CCK2Rs (K) and GPR173 (L) determined by the β-arrestin2 recruitment and β-arrestin1/2 recruitment assays (at least n = 3 independent experiments; one-way ANOVA with Tukey’s post hoc test or unpaired Student’s t test, ****p < 0.0001). Data are expressed as mean ± SEM.
As a low endogenous level of CCK1R/2Rs may be expressed in CHO-GPR173 cells, the CCK2R-specific agonist CCK4 (Lin et al., 1989) and CCK1R-specific agonist A-71 623 (Lin et al., 1991) were applied to exclude false-positive signals. As expected, CHO-CCK2R and CHO-CCK1R cells responded to CCK4 and A-71 623, respectively, in a concentration-dependent manner, but neither could induce Ca2+ release in CHO-GPR173 cells (Fig. 6D,E). We then tried to knock down the expression of GPR173 by applying shRNA (Gpr173) in CHO-GPR173 cells (Fig. 6F). The Ca2+ signal was downregulated by >50% after the mRNA level of GPR173 was knocked down by >50% (Fig. 6G). GPR173, also known as SREB3, is highly evolutionarily conserved and predominantly expressed in the CNS (Matsumoto et al., 2005; Regard et al., 2008). The endogenous ligands and physiological functions of GPR173 have not properly and directly been determined in the brain. A previous study of the phoenixin (PNX)/GPR173 system illustrates that PNX is essential for regulating the mRNA of GPR173 in the pituitary (Treen et al., 2016). However, from our cell-based assays, the CCK ligand CCK8s binds to GPR173 with nm potency. In contrast, PNX did not directly trigger an immediate Ca2+ response (Fig. 6H) or β-arrestin recruitment (Vandevoorde et al., 2018). Thus, PNX may modulate GPR173 activity or expression through an unknown pathway that needs further investigation.
These results indicate that the CCK8s-induced Ca2+ response in CHO-GPR173 cells was mediated by GPR173, again supporting that GPR173 is a novel CCK receptor.
GPR173 recruits β-arrestin after activation by CCK8s
CCK1R was reported to recruit β-arrestin1 (Ning et al., 2015). CCK2R was reported to recruit both β-arrestin1 and β-arrestin2 (Magnan et al., 2011, 2013). Of the four forms of arrestins (arrestin1, arrestin2, arrestin3, and arrestin4), arrestin2 and arrestin3, also named β-arrestin1 and β-arrestin2, respectively, are expressed in the brain (Gurevich and Gurevich, 2006; Latapy and Beaulieu, 2013).
In addition to the cell surface binding and Ca2+ imaging assays, we also applied a β-arrestin2 recruitment assay (PRESTO-Tango assay; Kroeze et al., 2015) to verify whether GPR173 is a CCK receptor. However, no β-arrestin2 recruitment was found for GPR173 after incubation with CCK8s (Fig. 6L). Consequently, we coexpressed β-arrestin1–conjugated TEV protease in the β-arrestin2 recruitment assay to detect whether GPR173 could recruit β-arrestins. We named this method the β-arrestin1/2 recruitment assay (Fig. 6I). We found that the β-arrestin1/2 recruitment signal for CCK1R was much higher than that in the β-arrestin2 assay (Fig. 6J), which is in line with previous reports that CCK1R could recruit β-arrestin1 (Ning et al., 2015). Meanwhile, the β-arrestin1/2 recruitment efficiency of CCK2R was far more than that in the β-arrestin2 recruitment assay (Fig. 6K), indicating that our modified β-arrestin recruitment system is reliable and more efficient. Next, we checked the dose-dependent response of CCK8s-induced β-arrestin recruitment for GPR173. We found that GPR173 responded to CCK8s in the β-arrestin1/2 recruitment assay (Fig. 6L).
In summary, CCK8s-induced β-arrestin recruitment further supports that GPR173 is likely a novel CCK receptor.
GPR173 is necessary for HFLS-induced iLTP
Considering the homology between CCK1R/2R and GPR173, we hypothesized that some reported CCK1R/2R antagonists might act on GPR173 and block CCK8s-induced Ca2+ responses. Thus, we selected several well-known CCK1R/2R antagonists that might inhibit CCK8s-induced Ca2+ responses in GPR173-CHO cells, including CCK1R antagonists devazepide (Bauer et al., 2018) and loxiglumide (Beglinger et al., 2001), and CCK2R antagonists YF476 (Mohammed et al., 2019) and L365,260 (Wu et al., 2014). Among these CCK1R/2R antagonists, devazepide had the highest antagonistic action on GPR173 with an IC50 of 12.3 nm for CCK8s-induced Ca2+ responses (Fig. 7A). Then, we examined whether devazepide could block HFLS-induced potentiation of inhibition in brain slices from mDlx-DIO-ChR2-mCherry-injected CCK-Cre mice using a patch-clamp recording protocol (Fig. 7B). We selectively patched pyramidal neurons in the virus-injected area of the AC (Fig. 7C) and found that the application of devazepide (60 nm in bath solution) fully blocked the HFLS-induced potentiation of IPSCs (Fig. 7D; individual and averaged traces of IPSCs; Fig. 7E; group data, 100.18 ± 1.91% vs 103.24 ± 2.54%, baseline vs 20–25 min after devazepide infusion with HFLS, two-way ANOVA with Tukey HSD, ****p < 0.0001, n = 8), in contrast with HFLS without application of devazepide (HFLS vs devazepide plus HFLS, 183.70 ± 6.25%, n = 9, vs 103.24 ± 2.54%, n = 8, two-way RM ANOVA, post hoc with Tukey HSD, ****p < 0.0001). The Ca2+ and patch-clamp assay also suggest that devazepide is an antagonist for GPR173. Although devazepide antagonizes both CCK1R and GPR173, the blockade of iLTP occurred through its antagonism of GPR173, as iLTP was not shown to be mediated by CCK1R or CCK2R (Fig. 3).
Devazepide as a GPR173 antagonist could block HFS-induced iLTP. A, The dose-dependent response curve of CHO-GPR173 cells activated by 10 nm CCK8s after incubation with antagonists for 30 min (at least n = 3 independent experiments). B, Injection of rAAV9-mDlx-DIO-ChR2-mCherry into CCK-Cre and PV-Cre mice. C, Schematic drawing shows in vitro patch-clamp recording from a target PC neuron while delivering a laser pulse to activate infected CCK neurons (top). D, Traces of IPSC responses to HFLS in brain slices from CCK-ires-Cre mice before and after HFLS (red) or devazepide + HFLS (black). E, Time course of normalized IPSCs before (for 5 min) and after (for 25 min) HFLS in CCK-ires-Cre mice or after (for 25 min) devazepide plus HFLS in CCK-Cre mice (black, n = 9 neurons; one-way ANOVA with Tukey’s post hoc test, N.S. not significant; two-way RM ANOVA with Tukey’s post hoc test, ****p < 0.0001). Data are expressed as mean ± SEM.
In summary, this evidence further supports that GPR173, a novel CCK receptor, mediates the CCK signal from CCK-INs and induces iLTP in their synapses.
Discussion
Because of the complexity of inhibitory neurons and connections, heterogeneous forms of inhibitory plasticity exist. Previous investigators proposed mechanisms for enhanced inhibitory control expressed at either the presynaptic or postsynaptic level. Different mechanisms of inhibitory plasticity at the presynaptic level that have been proposed include the release of more GABA transmitters (Caillard et al., 1999; Fiszman et al., 2005; Nugent et al., 2007), increased PV expression (Patz et al., 2004), and a GABAergic inhibition-induced decrease in glutamate release (Pan et al., 2009). iLTP is induced by postsynaptic activation of VGCCs (Kang et al., 1998; Caillard et al., 1999; Udakis et al., 2020). VGCCs provide a Ca2+ source for activating CaMKII (Udakis et al., 2020), which induces GABAAR phosphorylation and leads to iLTP (Petrini et al., 2014; He et al., 2015; Chiu et al., 2018). A study on awake mice identified a group of GABAergic neurons that burst or fire vigorously during rest and sharp-wave ripples and stay silent during locomotion and theta states (Szabo et al., 2022). CCK-INs are most active when the animal is at rest, whereas PV-INs remain silent (Dudok et al., 2021). However, most of these studies focus on inhibition-regulating behaviors depending on the hippocampus. More detailed mechanisms of iLTP from in vivo or long-term behavioral experiments remain elusive. Excitatory input arrives at the cell together with an inhibitory counterpart when an external stimulus activates the local cortex, which is normally recognized as the foundation of balanced cortical circuits (Galindo-Leon et al., 2009; Vogels et al., 2011; Alluri et al., 2016). We found that high-frequency activation of GABAergic neurons potentiated their long-term inhibition toward their target neurons and enhanced the general inhibition of the surrounding region, as reflected in the integrated neuronal responses to the forthcoming AS (Fig. 1). This iLTP in a the brain of a mature animal is critical for imbalanced excitation- and inhibition-induced brain disorders in adults (Vogels et al., 2011).
Considering the diverse electrophysiological, molecular, and structural features of GABAergic neurons, functional and behavioral correlates of this diversity are to be clarified accordingly. For example, PV-INs take leading control of the fast and powerful inhibitory effect on local neural activity, which makes them vital for the synchronization of spikes and synchronous gamma (30–80 Hz) oscillations (Kvitsiani et al., 2013; Chen et al., 2017; Jang et al., 2020). Stronger synchronization of PV-IN activity acts as a functional unit in the local medial prefrontal cortex, promoting goal-directed behavior (Kim et al., 2016). SOM-INs inhibit distal dendrites of excitatory neurons, causing them to act as key regulators of whisker movements in the somatosensory cortex (Yu et al., 2019) and the acquisition of motor skills in the motor cortex (Chen et al., 2015). Dendritic iLTP of SOM-IN synapses is driven by Ca2+ influx through NMDARs, whereas inputs from PV-INs and VIP-INs are unaffected (Chiu et al., 2018). These findings indicate the possibility of circuit-specific plasticity of GABAergic neurons.
In the visual cortex, high-frequency electrical stimulus trains (50 Hz) induce GABAAR-dependent LTP of inhibitory postsynaptic potentials of pyramidal neurons in developing rats (Komatsu, 1994). HFS of the subthalamic nucleus increases extracellular striatal GABA in hemiparkinsonian rats, which may benefit patients suffering from severe parkinsonism (Bruet et al., 2003). In the ventral tegmental area, HFS also potentiates IPSCs of GABAergic synapses, and this heterosynaptic LTP is NMDA receptor and GABAAR dependent (Nugent et al., 2007). In these previous studies, LTP was induced by electrical HFS, which activates all types of excitatory and inhibitory neurons and passing fibers and terminals. Here, we targeted only GABAergic neurons, specifically CCK-INs, and applied HFLS to investigate their postsynaptic connectivity with pyramidal cells using the whole-cell recording. We clearly demonstrate that iLTP of GABAergic output to pyramidal neurons was induced after HFLS of GABAergic neurons (Fig. 2). Our study focused on CCK-GABAergic output to pyramidal neurons. As CCK is colocalized in some VIP neurons (Gonchar, 2008), Lee et al. (2013) claim that some VIP-INs preferentially inhibit SOM-INs, increasing their activity during whisking and leading to disinhibitory control in the cortex by inhibiting SOM-INs or PV-INs (Pfeffer et al., 2013; Pi et al., 2013). Whether and how iLTP occurs among CCK-INs and other GABAergic neurons remains to be investigated.
CCK-GABAergic synapses modulate GABA release to the target PCs via presynaptic type 1 cannabinoid receptor (CB1R; Lee et al., 2010). The inability to potentiate inhibition to a forthcoming AS after HFLS of local GABAergic neurons in Vgat-Cre-CCK-cKO mice suggests that CCK is crucial for iLTP (Fig. 1), although this observation is because of less CCK-induced decreased GABA release via presynaptic GPR173 or CB1R, which requires further study. CCK-INs directly project to the soma of pyramidal cells (Fig. 5G; Kawaguchi and Kubota, 1998), supporting our observation that IPSCs (with a short latency of 4.53 ± 0.28 ms, n = 10 cells) of pyramidal cells induced by optogenetically activating CCK-INs is a directly induced response rather than a triggered response of crossing mutineuron nodes (Fig. 2). The significant potentiation of IPSCs caused by HFLS of CCK-INs and the induction of iLTP in the presence of exogenous CCK8s after LFLS of CCK-INs further validates the critical role of CCK in iLTP induction (Fig. 2). Moreover, the anxiolytic effect of CCK-INs in the ventral hippocampus of rats (Moghaddam et al., 2012) and the observation that CCK-IN activity inversely scales with pyramidal cell activity (Dudok et al., 2021) suggests that CCK-INs also play a functional role in the hippocampus. How CCK-INs switch inhibitory plasticity in other brain areas and how they interact with other GABAergic neurons in inhibitory plasticity remain to be explored.
CCK1R and CCK2R were discovered in the brain several decades ago (Van Dijk et al., 1984; Hill et al., 1987), which was followed by investigations of their gene expression (Honda et al., 1993) and associated behaviors and disorders (Reubi et al., 1997; Horinouchi et al., 2004; Cayrol et al., 2006). Later research mainly focused on the signaling, biological activities, and structure of these two characterized CCK receptors (Ning et al., 2015; Zeng et al., 2020; Ding et al., 2022), with less attention paid to identifying new receptors. The induction of iLTP in CCK1R/2R-KO mice after HFLS of CCK-INs (Fig. 3) suggests that an unknown CCK receptor (i.e., CCK3R) mediates this synaptic plasticity. Because of receptor expression levels, the probing efficiency of ligands, and the readout of autoradiographic experiments, the possibility remains that unknown receptors with low expression and selective binding potency with specific isoforms of CCK were missed during our characterization. Based on our in vivo and in vitro recording experiments, we believe that new CCK receptors are awaiting clarification, which will significantly challenge our current understanding. Using different bioinformatics algorithms, we narrowed the range of CCK receptor candidates. We demonstrated that GPR173 is a novel CCK receptor by histology and three independent cell-based assays (Figs. 4–6). CCK8s-induced iLTP in cortical slice recordings (Fig. 2), Ca2+ mobilization, and recruitment of β-arrestin in the cell line assay (Fig. 6) possibly occur through binding and activation of GPR173. GPR173 receptors are expressed specifically in excitatory neurons rather than inhibitory cells and are localized in CCK-GABAergic synapses in the AC (Fig. 5E,F). A GPR173 antagonist, devazepide, entirely blocked HFLS-induced iLTP, confirming the role of GPR173 in iLTP induction (Fig. 7).
Combining the current understanding with our new finding of CCK3R, we propose several possible mechanisms for iLTP induction. The activation of CCK3R by CCK release leads to Ca2+ signaling, possibly through the opening of Ca2+ channels in postsynaptic neurons or the release of Ca2+ from the endoplasmic reticulum. iLTP may also require an increase of gephyrin and postsynaptic GABAARs and CaMKII activation, as in previous studies (Petrini et al., 2014; He et al., 2015; Chiu et al., 2018; Udakis et al., 2020). Our cell-based assays indicate CCK application triggers a Ca2+ signal. Ca2+ signaling potentially enables GPR173-mediated iLTP via the following mechanisms: (1) by increasing GABAAR insertion in the postsynaptic surface membrane through promoting exocytosis (Petrini et al., 2014), (2) by increasing mobilization of GABAARs in the postsynaptic cell, or (3) by stabilizing GABAAR or VGCC gating properties. Similar to glutamatergic synapses, GABAergic synapses undergo homosynaptic and heterosynaptic plasticity (Ravasenga et al., 2022). In the current study, we could not exclude the possibility that CCK-IN–induced iLTP is restricted to the CCK postsynaptic area without affecting neighboring PV-IN synapses or other synapses. Whether this GPR173-mediated iLTP is homosynaptic or heterosynaptic remains to be investigated in the future.
In summary, we demonstrated that HFLS of local GABAergic neurons induced iLTP, which was CCK dependent. We report a novel CCK receptor, GPR173, localized in CCK-GABAergic synapses that mediates CCK-mediated iLTP. The involvement of CCK and its new receptor CCK3R in iLTP indicates their crucial roles in enhancing the inhibitory level in the neocortex and possibly other regions, allowing maintenance of the excitation and inhibition balance of the brain. As CCK3R mediates the potentiation of iLTP rather than directly inhibits neuronal activity, it may provide a better target for treating brain disorders related to excitation/inhibition balance, such as epilepsy, schizophrenia, and depression.
