{'fraction_non_alphanumeric': 0.05465313028764805, 'fraction_numerical': 0.038612521150592216, 'mean_word_length': 4.652448355011477, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 1, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Neuropeptide S Receptor Stimulation Excites Principal Neurons in Murine Basolateral Amygdala through a Calcium-Dependent Decrease in Membrane Potassium Conductance

Background: The neuropeptide S system, consisting of the 20 amino acid neuropeptide NPS and its G-protein-coupled receptor (GPCR) neuropeptide S receptor 1 (NPSR1), has been studied intensively in rodents. Although there is a lot of data retrieved from behavioral studies using pharmacology or genetic interventions, little is known about intracellular signaling cascades in neurons endogenously expressing the NPSR1. Methods: To elucidate possible G-protein-dependent signaling and effector systems, we performed whole-cell patch-clamp recordings on principal neurons of the anterior basolateral amygdala of mice. We used pharmacological interventions to characterize the NPSR1-mediated current induced by NPS application. Results: Application of NPS reliably evokes inward-directed currents in amygdalar neurons recorded in brain slice preparations of male and female mice. The NPSR1-mediated current had a reversal potential near the potassium reversal potential (EK) and was accompanied by an increase in membrane input resistance. GDP-β-S and BAPTA, but neither adenylyl cyclase inhibition nor 8-Br-cAMP, abolished the current. Intracellular tetraethylammonium or 4-aminopyridine reduced the NPS-evoked current. Conclusion: NPSR1 activation in amygdalar neurons inhibits voltage-gated potassium (K+) channels, most likely members of the delayed rectifier family. Intracellularly, Gαq signaling and calcium ions seem to be mandatory for the observed current and increased neuronal excitability.


Introduction
The neuropeptide S system, consisting of the 20 amino acid neuropeptide NPS and its G-protein-coupled receptor (GPCR) neuropeptide S receptor 1 (NPSR1) has been identified in the central nervous system of rodents and humans [1,2]. NPS-expressing neurons seem to be largely restricted to distinct brain stem nuclei, located in the pericoerulear region and between the lateral parabrachial and Koelliker-Fuse nucleus in mice [3,4]. In contrast, NPSR1-expressing neurons are found in a variety of regions within the central nervous system of rodents, e.g., olfactory areas, amygdala, frontal and retrosplenial cortex, and midline thalamic regions [3]. Pharmacological studies and use of NPS-or NPSR1-deficient mice point to an important role of the NPS system, e.g., in memory formation [5,6], fear and anxiety [7][8][9][10], social behavior [11], addiction [12,13], and arousal/attention [1,5].
GPCR-mediated signaling can be G protein dependent or, according to more recent concepts, independent [14]. G proteins can be subdivided into four major families: G q/11 , G S , G i/o , or G 12/13 , all triggering different intracellular signaling cascades when activated [15]. Upon activation, the heterotrimeric G protein dissociates into an α and a β/γ subunit, each able to modulate intracellular or membrane-located effectors. G protein 2 of 17 signaling is initiated by the exchange of GDP by GTP, and is terminated when GTP is hydrolyzed to GDP by the α subunit [15]. In addition to G proteins, activated GPCRs can couple to arrestins or G-protein-coupled receptor kinases (GRK), which, in turn, can contribute to intracellular signaling cascades or lead to GPCR desensitization and internalization.
Early studies in HEK and CHO cell lines expressing human or murine NPSR1 showed that NPSR1 activation by its ligand triggers intracellular signaling cascades involving an increase in intracellular calcium levels, formation of cAMP, and MAPK phosphorylation [16][17][18]. These findings strongly suggest that NPSR1 activation is followed by signaling cascades involving G q and G s . In addition, calcium transients following NPSR1 activation have been observed in neurons, and pharmacological interventions strongly suggest pathways dependent on phospholipase C (PLC), inositol-3-phosphate receptors (IP 3 R), and ryanodine receptors (RyR) to be involved in NPSR1 signaling [19][20][21].
Here we used a straight-forward electrophysiological approach to identify the ionic nature of the NPSR1-mediated current in principal neurons of the anterior basolateral amygdala (aBA PNs), which have been implicated in processes of fear and extinction [24]. We identified putative intracellular signaling pathways following NPSR1 activation, consequently inducing a reduction of K + conductances, and thereby increasing neuronal discharges.

NPS-Induced Inward Currents in NPSR1-Expressing aBA Principal Neurons
As described previously [3,8,24], we detected NPSR1-coding mRNA predominantly in the anterior part of the basolateral amygdala (aBA) by fluorescence in situ hybridization in mouse brain slices ( Figure 1A). A second population of NPSR1-expressing principal neurons (PN) is located in the lateral nucleus of the amygdala (LA; [24]), but the posterior regions of the basolateral nucleus and the central nucleus of the amygdala (CeA) are largely devoid of NPSR1 mRNA. We performed whole-cell voltage-clamp recordings from PNs in coronal slices containing the aBA ( Figure 1B) to analyze intracellular signaling cascades following NPSR1 activation by NPS. Application of 50 or 150 nM NPS elicited a transient inward current in aBA PNs ( Figure 1C). The overall fraction of NPS-responsive neurons was 85.7 and 91.5 % following 50 or 150 nM NPS, respectively. The current density evoked by 150 nM was significantly larger compared to current densities evoked by 50 nM (150 nM NPS: −0.70 ± 0.09 pA/pF; 50 nM NPS: −0.42 ± 0.04 pA/pF; RM-ANOVA interaction of time and concentration: F(2,38) = 6.8; p = 0.003; Bonferroni post hoc: 150 baseline vs. 150 max: p = 1.3 × 10 −11 ; 50 baseline vs. 50 max: p = 1.3 × 10 −5 ; 150 max vs. 50 max: p = 0.032; Figure 1D,E). Since male and female mice were used, we analyzed the current density in both sexes in a separate set of experiments ( Figure 1F,G). Neither the time course nor the maximal current density differed between male and female mice (males: −0.657 ± 0.09 pA/pF; n = 11; females: −0.656 ± 0.05 pA/pF; n = 8; t-test: t = −0.09; df = 17; p = 0.926). In the following, data from both sexes were pooled. During voltage-clamp recordings, the input resistance of the cell was monitored by applying brief voltage steps (−5 mV; 50 ms duration) in each recorded sweep (10 s duration). Under baseline conditions, the input resistance was 167 ± 6 MΩ, and increased significantly in the presence of 150 nM NPS to 208 ± 8 MΩ (paired t-test: t = −7.82; df = 36; p = 2.8 × 10 −9 ; n = 37). voltage-clamp recordings, the input resistance of the cell was monitored by applying brief voltage steps (-5 mV; 50 ms duration) in each recorded sweep (10 s duration). Under baseline conditions, the input resistance was 167 ± 6 MΩ, and increased significantly in the presence of 150 nM NPS to 208 ± 8 MΩ (paired t-test: t = −7.82; df = 36; p = 2.8 × 10 −9 ; n = 37). To test the specificity of our approach, we applied NPS in the presence of the NPSR1-specific antagonist SHA-68 ( [25]; Figure 1H). The fraction of non-responsive neurons significantly increased to 57 % in the presence of SHA-68, compared to 6 % un- To test the specificity of our approach, we applied NPS in the presence of the NPSR1specific antagonist SHA-68 ( [25]; Figure 1H). The fraction of non-responsive neurons significantly increased to 57 % in the presence of SHA-68, compared to 6 % under control conditions (Fisher's exact test: p = 0.012; Figure 1I). Only three of the recorded aBA PNs showed a detectable inward current upon NPS application, which was significantly decreased compared to controls (control: −0.679 ± 0.07 pA/pF; n = 17; SHA-68: −0.116 ± 0.032; n = 3; t-test: t = −3.28; df = 18; p = 0.004; Figure 1J,K). These data substantiate the evidence that NPSR1 activation by NPS induces a transient inward-directed current in aBA PNs of male and female mice. In this way, NPS could excite the neurons and activate amygdala networks.

The NPSR1-Dependent Current Results from Reduced Potassium Conductances
To characterize the nature of the NPSR1-mediated current, hyper-and depolarizing voltage steps with increasing amplitudes were applied from a holding potential of −60 mV (Figure 2A). The NPS-induced current was calculated at each voltage step by subtraction of the current obtained during baseline. Amplitudes of the instantaneous current at the beginning and the steady state current at termination of the voltage step were analyzed, and the calculated NPS-induced current was plotted against the respective step potential ( Figure 2B). The reversal potential of the NPSR1-mediated current was −101.5 ± 0.8 mV (n = 4) for steady state currents and, thus, was close to the calculated K + reversal potential (E K ) of −109 mV. To confirm these findings, hyperpolarizing voltage-clamp ramps were performed from 0 mV to −120 mV in 250 ms ( Figure 2C). The NPS-induced current calculated by subtraction of ramp NPS -ramp baseline had a reversal potential of −101.2 ± 2.8 mV (n = 3), again close to E K ( Figure 2D). Therefore, we reasoned that the NPSR1-mediated inward current is due to a reduction of a K + conductance. Therefore, reducing the electrochemical gradient for K + should affect the amplitude of the NPSR1-mediated current recorded at −60 mV. We shifted E K from −109 mV to −73.5 mV by increasing extracellular K + from 2.5 to 10 mM, thereby decreasing the electrochemical driving force from approximately −49 mV to −13.5 mV. The current density of the NPS-induced current was reduced to −0.23 ± 0.05 pA/pF (n = 6) in 10 mM K + , and was significantly smaller than under control conditions (control: −0.668 ± 0.06 pA/pF; n = 15; t-test: t = −4.31; df = 19; p = 3.8 × 10 −4 ; Figure 2E,F).  Figure 1I). Only three of the recorded aBA PNs showed a detectable inward current upon NPS application, which was significantly decreased compared to controls (control: −0.679 ± 0.07 pA/pF; n = 17; SHA-68: −0.116 ± 0.032; n = 3; t-test: t = −3.28; df = 18; p = 0.004; Figure 1J,K). These data substantiate the evidence that NPSR1 activation by NPS induces a transient inward-directed current in aBA PNs of male and female mice. In this way, NPS could excite the neurons and activate amygdala networks.

The NPSR1-Dependent Current Results from Reduced Potassium Conductances
To characterize the nature of the NPSR1-mediated current, hyper-and depolarizing voltage steps with increasing amplitudes were applied from a holding potential of −60 mV (Figure 2A). The NPS-induced current was calculated at each voltage step by subtraction of the current obtained during baseline. Amplitudes of the instantaneous current at the beginning and the steady state current at termination of the voltage step were analyzed, and the calculated NPS-induced current was plotted against the respective step potential ( Figure 2B). The reversal potential of the NPSR1-mediated current was −101.5 ± 0.8 mV (n = 4) for steady state currents and, thus, was close to the calculated K + reversal potential (EK) of −109 mV. To confirm these findings, hyperpolarizing voltage-clamp ramps were performed from 0 mV to −120 mV in 250 ms ( Figure 2C). The NPS-induced current calculated by subtraction of rampNPS-rampbaseline had a reversal potential of −101.2 ± 2.8 mV (n = 3), again close to EK ( Figure 2D). Therefore, we reasoned that the NPSR1-mediated inward current is due to a reduction of a K + conductance. Therefore, reducing the electrochemical gradient for K + should affect the amplitude of the NPSR1-mediated current recorded at −60 mV. We shifted EK from −109 mV to −73.5 mV by increasing extracellular K + from 2.5 to 10 mM, thereby decreasing the electrochemical driving force from approximately −49 mV to −13.5 mV. The current density of the NPS-induced current was reduced to −0.23 ± 0.05 pA/pF (n = 6) in 10 mM K + , and was significantly smaller than under control conditions (control: -0.668 ± 0.06 pA/pF; n = 15; t-test: t = −4.31; df = 19; p = 3.8 × 10 −4 ; Figure 2E,F).

The NPS-Induced Current Is Dependent on NPSR1-Gα-Signaling
Activation of the NPSR1 by its ligand NPS triggers intracellular signaling pathways via G αq and/or G αs proteins, as shown in HEK and CHO cells expressing the receptor [1,17,19]. In contrast, the contribution of G protein activity to the induction of the inward current observed here is still unknown. To test the significance of G protein signaling, voltageclamp recordings in the presence of 2 mM GDP-β-S were performed ( Figure 3A). GDP-β-S blocks the activation of G proteins by inhibiting binding of GTP to G proteins [26]. Adding 2 mM GDP-β-S to the intracellular solution prevented responses to 50 nM NPS in aBA PNs ( Figure 3B). Only one of six recorded neurons did show a detectable inward current ( Figure 3E). To quantify these findings, only here we averaged recorded currents of responsive and non-responsive neurons in the presence and absence of GDP-β-S. Statistical comparison revealed a significant reduction of mean current densities (RM ANOVA: interaction treatment x time: F(2,40) = 3.93; p = 0.028; Bonferroni post hoc test: NPS max control vs. NPS max GDP-β-S: p = 0.034; baseline control vs. NPS max control: p = 8.5 × 10 −7 ; baseline control vs. wash control: p = 0.006; Figure 3B,C) and a significantly reduced fraction of responsive neurons (Fisher's exact test: p = 0.005; Figure 3E). Application of 150 nM NPS induced a current in 86 % of the recorded neurons in the presence of GDP-β-S ( Figure 3E), while the mean current density was significantly reduced to −0.08 ± 0.04 pA/pF in these neurons (n = 6; Figure 3H). Next, we used gallein (50 µM) to inhibit G βγ signaling in aBA PNs during application of 150 nM NPS ( Figure 3F). Although a trend was observed, the mean current densities were not significantly reduced in the presence of gallein (gallein: −0.48 ± 0.07 pA/pF; n = 10; control: −0.667 ± 0.07; n = 17; one-way ANOVA: F(2,30) = 9.71; p= 5.5×10 −4 ; Bonferroni post hoc test: control vs. GDP-β-S: p = 4.1 × 10 −4 ; gallein vs. GDP-β-S: p = 0.047; Figure 3G,H). These data strongly suggest that mostly G α signaling following NPSR1 activation in aBA PNs mediates the NPS-induced current.

Pharmacological Characterization of the NPSR1-Modulated K + Conductance
In the next series of experiments, we further characterized the K + conductance evoked by NPSR1 stimulation. We applied tetraethyl-ammonium (10 mM) via the ACSF to inhibit a broad spectrum of delayed-rectifier K + channels, XE991 (20 µM) to inhibit Mcurrents generated by members of the KCNQ channel family, and BaCl2 (0.5 mM) to inhibit classes of inward-rectifier and G-protein-dependent inward-rectifier (G irk ) K + channels ( Figure 5A,C). None of these extracellularly applied compounds inhibited the NPSR1mediated current upon application of 150 nM NPS. However, using a cesium-based intracellular solution containing 10 mM TEA significantly reduced the NPS-elicited current (Cs intra : −0.257 ± 0.04 pA/pF; n = 8; control: −0.703 ± 0.09 pA/pF; n = 11; Figure 5B,C). To differentiate between inhibition of K + channels by cesium or intracellular TEA, we used a K + -gluconate-based recording solution containing 10 mM TEA. Intracellular TEA alone reduced the NPSR1-mediated current significantly (K-gluc-TEA intra : −0.264 ± 0.04 pA/pF; n = 16; control: −0.703 ± 0.09 pA/pF; n = 11; Figure 5B,C).

NPSR1 Activation Enhances Action Potential Generation in aBA PNs
NPSR1-dependent reduction of K + conductances likely affects the excitability of aBA PNs. To test this possibility, we performed current-clamp recordings during baseline conditions and during near-maximal NPS effects ( Figure 6A). Passive and active membrane properties were analyzed by injecting hyper-and depolarizing rectangular current pulses (500 ms duration), with step-wise (+20 pA) increases in amplitude from a membrane potential of either −80 or −60 mV. The mean resting membrane potential of aBA PNs was at −75 ± 1 mV, and the mean membrane capacitance was 64 ± 5 pF (n = 14). Next, hyperpolarizing currents were injected from a membrane potential of −80 mV. The input resistance during control and in the presence of NPS was calculated from the slope of the I-V plots (R in = ∆U/∆I). Although the change of the membrane potential in response to hyperpolarizing current injections from −80 mV was not significantly different in the presence and absence of NPS ( Figure 6B), the calculated input resistance was significantly enhanced in the presence of NPS. R in was 132 ± 12 MΩ during control conditions, and increased to 153 ± 14 MΩ in the presence of NPS (paired t-test: t = −4.56; df = 9; p = 0.001). In addition, action potential generation in response to depolarizing currents was enhanced in the presence of NPS (RM-ANOVA: interaction treatment x current F(6108) = 4.87; p = 1.9 × 10 −4 ; LSD post hoc test: baseline vs. NPS 120 pA: p = 0.042; 140 pA p = 0.035; 160 pA p = 0.006; 180 pA p = 0.002; n = 10; Figure 6C). Concomitantly, we observed a negative shift of the action potential threshold during NPSR1 application, while other active membrane properties were unchanged (threshold control vs. threshold NPS : paired t-test: t = 2.63; df = 9; p = 0.027; n = 10; Figure 6F). Furthermore, we observed significant effects of NPS application for hyperpolarizing current injections at −60 mV. Larger changes of the membrane potential in the presence of NPS were found, thereby indicating an increase in the input resistance (RM-ANOVA interaction treatment x current F(5,130) = 4.29; p = 0.001; LSD post hoc test: baseline vs. NPS: −100 pA p = 0.01; −80 pA p = 0.003; −60 pA p = 0.021; −40 pA p = 0.01; n = 14; Figure 6D). The mean input resistance calculated from the I-V plots was 192 ± 12 MΩ during baseline conditions, and increased to 268 ± 24 MΩ in the presence of NPS (paired t-test: t = −3.061; df = 13; p = 0.009; n = 14). In contrast, NPS failed to significantly increase neuronal discharges in response to depolarizing current injections at -60 mV (RM-ANOVA: treatment vs. current F(7175) = 1.25; p = 0.28; n = 14; Figure 6E). These data show that NPSR1 activation enhances neuronal discharges, accompanied by a reduction of the action potential threshold and a moderate increase of input resistance. 0.001). In addition, action potential generation in response to depolarizing currents was enhanced in the presence of NPS (RM-ANOVA: interaction treatment x current F(6108) = 4.87; p = 1.9 × 10 −4 ; LSD post hoc test: baseline vs. NPS 120 pA: p = 0.042; 140 pA p = 0.035; 160 pA p = 0.006; 180 pA p = 0.002; n = 10; Figure 6C). Concomitantly, we observed a negative shift of the action potential threshold during NPSR1 application, while other active membrane properties were unchanged (thresholdcontrol vs. thresholdNPS: paired t-test: t = 2.63; df = 9; p = 0.027; n = 10; Figure 6F).

Discussion
The vast majority of available studies describe the function of the NPS system on a network level [8,22], analyze behavioral consequences following pharmacological or genetic interventions [5,9,31], or point out implications of NPSR1 single-nucleotide polymorphisms for psychiatric disorders in humans [32,33]. Other studies analyzed structure-function relationships of NPSR1 and NPS, using HEK or CHO expression systems and rodent models [24,34,35]. In contrast, little is known about signaling cascades in neurons endogenously expressing the NPSR1.
Here, we used electrophysiological approaches, combined with pharmacology, to investigate intracellular signaling and possible second messengers and effectors in principal neurons of the anterior basolateral complex of mice. Based on our data, we can (a) confirm the expression profile of NPSR1 mRNA in the aBA, (b) describe NPS-specific activation of the NPSR1 in aBA PNs of male and female mice, (c) provide evidence that the NPSR1mediated inward current is dependent on Gαq signaling, (d) identify a reduction of membrane K + conductance in these neurons as consequence of NPSR1 stimulation, and (e) show NPS-induced increase in neuronal excitability.
As described previously [3,8,24], high NPSR1 mRNA expression can be detected in the aBA of mice, whereas it is almost absent in the posterior BA or the central nuclei of the amygdala. NPSR1 activation by the application of NPS induced a transient inward current in about 80 to 90 % of the recorded aBA PNs, which was abolished by the NPSR1-specific antagonist SHA-68 [25]. The NPSR1-mediated current was not different between males and females, in line with our previous findings documenting a lack of sex-based differences on the cellular level [24]. This inward current was accompanied by an increase in apparent input resistance, which has also been described in projection neurons of the endopiriform nucleus [22]. These findings indicate a reduction of membrane conductance, which is active at a membrane potential of −60 mV. Analysis of current-voltage relationships of the NPS-induced current and recordings in elevated extracellular K + concentrations revealed a reversal potential near the respective K + equilibrium potential, identifying K + ions as carriers of the current. GPCRs can modulate K + channel activity through changes in channel conductance and/or open probability. For example, activation of muscarinic receptors induces G-protein-mediated negative modulation of K2P channels involving different intracellular signaling molecules [36,37] in, e.g., thalamic relay neurons.
The NPSR1-mediated current requires intracellular G protein activity, as inhibition by GDP-β-S abolished the inward current. Thus, direct protein-protein interaction of the NPSR1 and a putative effector seems not to be involved. Furthermore, inhibition of the β/γ-subunits by gallein had no effect on the NPSR1-mediated current, leading to the conclusion that Gα-signaling is mandatory. Depending on the α-subunit involved (αs and/or αq), different downstream signaling molecules, such as adenylyl cyclases (AC; by G αs ) or phospholipases (PL; G αq ), would be activated in prototypic pathways. Here, NPSR1-mediated currents were insensitive to AC inhibition or intracellular cAMP application, ruling out a major contribution of Gαs signaling, which has been described to occur after NPSR1 activation in HEK or CHO cells [17,38]. In contrast, the NPSR1-mediated current in aBA PNs was strongly reduced by manipulation of intracellular Ca 2+ by either BAPTA or 2-APB. Intracellular Ca 2+ -transients have been described in detail previously in cultured hippocampal neurons expressing human NPSR1 [19]. In these assays, 2-APB abolished the rise of intracellular Ca 2+ , most likely due to inhibition of IP3Rs and SOCE. In addition, in dorsal raphe (DR) and laterodorsal tegmentum (LT), NPS application induced an increase of intracellular Ca 2+ involving IP3Rs and RyR [21]. Moreover, the NPSR1mediated current was also dependent on Ca 2+ in DR and LT neurons. Thus, mobilization of intracellular Ca 2+ as a second messenger seems to be shared by aBA PNs and DR/LT neurons following NPSR1 activation. It is interesting to note that Ca 2+ release induced by PLC activation feeds back on PLC, a strong positive feedback mechanism that is sensitive to BAPTA application [39], thereby emphasizing the significance of the G αq /PLC pathway for the present findings.
While the involvement of K + channels seems to be clear, our pharmacological assays provide some hints with respect to K + channel isoforms modulated by NPSR1, without identifying the exact types of channels involved. Results obtained with barium at the concentration used in the present study exclude the involvement of G-protein-coupled inward rectifier (G irk ) channels as potential targets. The concentration of XE991 used inhibits KV7.1/2/4, but not KV7.5, which has been shown to mediate M-type currents in various neurons of various brain regions, including BLA [40]. Overall, the NPS-evoked K + current was sensitive to intracellular TEA and 4-AP, typifying delayed rectifier and A-type K + channels. 4-AP-sensitive K + channels of the KCND family mediate a transient, hyperpolarization-dependent A-type current, which is thought to mediate a delayed onset of spike firing if evoked from membrane potentials negative from resting membrane poten-tials [41]. The lack of noticeable delayed onset of firing in our current-clamp recordings from −80 mV suggests that A-type currents are not prominently present in aBA PNs, and are unlikely to mediate the NPSR1-mediated current. The lack of effect of THA or PK-THPP excludes TASK channels as targets of NPSR1 signaling [42]. Taken together, these data leave members of the delayed rectifier classes and Kv7.5 as most plausible candidates. M-current mediating K V 7.5 channels are inhibited directly by intracellular Ca 2+ [43] or, e.g., by A-kinase-anchoring protein AKAP150 [44] following GPCR-activation. Thus, NPSR1dependent intracellular signaling cascades, including Ca 2+ , could negatively modulate K V 7.5 channels. Of note, we cannot completely rule out additional modulations of voltagegated Ca 2+ channels in aBA PNs by NPSR1 activation [45,46]. Future experiments using elaborated pharmacology and/or single-cell mRNA sequencing are needed to identify the exact target channels. Delayed rectifier channels of the Kv2.1 subtype, characterized by PLC-mediated regulation, 4-AP-sensitivity, and incomplete inactivation, are promising candidates [47,48].
In summary, we show that NPSR1 activation by NPS increases neuronal excitability in aBA PNs by the inhibition of voltage-gated K + channels. We provide data indicating the involvement of G αq -and intracellular calcium signaling in these neurons. As a functional consequence, NPS release in aBA will increase local network activity and can thereby modulate information processing in emotion-relevant circuits between aBA and the posterior BA (pBA), as shown before [24,49,50].
Thus, the NPS/NPSR1 system is apt to shape emotional states in response to aversive stimuli and to modulate threat responses during expression of learned fear and fear extinction [7,8,24]. In turn, alterations of NPSR1 signaling efficacy by nonsynonymous mutations in the NPSR1 [24,32,33,51] or the NPS gene [51] might consequently alter behavior and the risk for the development of psychiatric disorders. It is evident that detailed knowledge about neuropeptide systems and underlying signaling cascades is important to understand their impact on the development of psychiatric disorders. An increasing number of different neuropeptide systems (e.g., galanin, cholecystokinin, and NPY) have been shown to modulate fear [52][53][54][55] and anxiety [56][57][58] in rodents and humans [59]. In this respect, it is interesting to note that orexin, an excitatory neuropeptide with well-described roles in regulation of arousal and energy homeostasis, was recently found to modulate fear responses [60]. The pathway underlying in this modulation involved the PLC-dependent depolarization of neurons in the central nucleus of the amygdala, thereby pointing to some similarities in neuropeptide signaling pathways and the in vivo relevance of their modulatory influence. Therefore, detailed analysis of GPCR signaling, effects of humanrelevant polymorphisms in neuropeptide systems, and the interplay between different neuropeptide systems is needed to understand and possibly treat psychiatric diseases.

Materials and Methods
All animal experiments were carried out in accordance with European regulations on animal experimentation (European Committee Council Directive 2010/63/EU; National Research Council of the National Academies), and approved by the local authorities (LANUV).

Animals
C57BL/6N were kept in a temperature-controlled (21 • C) and humidity-controlled (50-60 % relative humidity) animal facility in individually ventilated cages, with access to food and water ad libitum and a 12 h light/dark cycle, with lights on at 6:00 am. Food, water, and animal conditions were controlled on a daily basis. For experiments, male and female mice 6 to 8 weeks of age were used.

Electrophysiology
Experimental procedures were carried out as described previously [24]. Mice were decapitated, and brains were quickly removed. Coronal or horizontal brain slices (300 µm thickness), containing the amygdala, were cut on a vibratome (VT1200S; Leica, Germany).
Voltage-clamp step protocols were performed from a holding potential of −60 mV, and steps from −120 mV to −50 mV (∆ + 10 mV) were applied for two seconds each. Resultant current traces during baseline and in the presence of NPS were subtracted (current NPScurrent baseline ) to calculate the NPS-induced (NPSR1-mediated) current. The reversal potential of the NPS-induced current was calculated for each individual PN. Hyperpolarizing voltageclamp ramps were done from a step to 0 mV (1 s) to −120 mV (ramp velocity: 0.48 mV/ms). The NPS-induced current was calculated from ramp NPS and ramp baseline , analyzed as described above. Substances for pharmacological interventions: n-[((4-fluorophenyl)methyl)tetrahydro-3-oxo-1,1-diphenyl-3H-oxazolo ( To inhibit adenylyl cyclases, slices were preincubated in ACSF containing SQ 22536 for >1 h, and SQ 22536 was included in the pipette during subsequent recordings. The concentration of the anorganic solvent dimethylsulfoxide (DMSO) was kept below 0.02 % when present. DMSO alone at the concentration used did not interfere with the observed NPSR1-mediated current.