Ionic signaling mechanisms involved in neurokinin-3 receptor-mediated augmentation of fear-potentiated startle response in the basolateral amygdala

Abstract

The tachykinin peptides include substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), which interact with three G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs, and NK3Rs, respectively. Whereas high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3Rs in this brain region have not been determined. We found that activation of NK3Rs by application of the selective agonist, senktide, persistently excited BLA principal neurons. NK3R-elicited excitation of BLA neurons was mediated by activation of a nonselective cation channel and depression of the inwardly rectifying K⁺ (Kir) channels. With selective channel blockers and knockout mice, we further showed that NK3R activation excited BLA neurons by depressing the G protein-activated inwardly rectifying K⁺ (GIRK) channels and activating TRPC4 and TRPC5 channels. The effects of NK3Rs required the functions of phospholipase Cβ (PLCβ), but were independent of intracellular Ca²⁺ release and protein kinase C. PLCβ-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) was involved in NK3R-induced excitation of BLA neurons. Microinjection of senktide into the BLA of rats augmented fear-potentiated startle (FPS) and this effect was blocked by prior injection of the selective NK3R antagonist, SB 218795, suggesting that activation of NK3Rs in the BLA increased FPS. We further showed that TRPC4/5 and GIRK channels were involved in NK3R-elicited facilitation of FPS. Our results provide a cellular and molecular mechanism whereby NK3R activation excites BLA neurons and enhances FPS.

Graphical Abstract

Neurokinin-B (NKB) is a member of the tachykinin family of peptides that act as a neurotransmitter or neuromodulator in central brain circuits and the amygdala is an important target for this peptide. NKB activates the neurokinin-3 receptors (NK3Rs) which are G_q/11-coupled receptors signaling through the PLCβ pathway. While high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3R activation in this brain region have not been determined. We found that activation of NK3Rs excited BLA principal neurons by activating TRPC4/5 channels and depressing the GIRK type of the inwardly rectifying K⁺ channels. PLCβ-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP₂) was responsible for NK3R-induced excitation of BLA neurons. Activation of NK3Rs in the BLA significantly increased the fear-potentiated startle responses via activation of TRPC4/5 channels and suppression of GIRK channels. Our results provide a cellular and molecular mechanism to explain NK3R-elicited augmentation of fear responses.

Introduction

The tachykinins refer to the peptides encoded in rodents by the Tachykinin 1 (Tac1) and Tac2 (TAC3 in humans) genes, which are involved in neurotransmission and neuromodulation in the central nervous system (Beaujouan et al., 2004). Tac1 encodes a precursor protein that produces two peptides, substance P (SP) and neurokinin A (NKA), whereas Tac2/TAC3 encodes neurokinin B (NKB). SP, NKA and NKB interact respectively with the G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs, and NK3Rs (Gerard et al., 1993; Maggi, 1995). These receptors are coupled to the pertussis toxin-insensitive G proteins G_q/11 to activate phospholipase Cβ (PLCβ) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP₂), resulting in the production of inositol 1,4,5-triphosphate (IP₃) to facilitate intracellular Ca²⁺ release and diacylglycerol (DAG) to activate protein kinase C (PKC) (Khawaja & Rogers, 1996; Steinhoff et al., 2014), although activation of adenylate cyclase, resulting in accumulation of cAMP and stimulation of protein kinase A and activation of phospholipase A2 and generation of arachidonic acid have been reported as well (Steinhoff et al., 2014). In mammals, tachykinins serve as neuromodulators or neurotransmitters in central brain circuits, as well as in pain, stress, anxiety, depressive disorder, aggression, memory formation, inflammation and hormone regulation (Onaga, 2014; Steinhoff et al., 2014; Lenard et al., 2018; Zieglgansberger, 2019). However, the cellular and molecular mechanisms underlying tachykinins-mediated modulation of these physiological functions and pathological disorders have not been fully determined.

The amygdala is a key region of the brain involved in processing and propagating fear- and anxiety-related signals. The amygdala is an assembly of nuclei comprising the lateral amygdala (LA), the basolateral amygdala (BLA), and the central amygdala (CeA). The CeA is formed by 3 subnuclei named as capsular, lateral, and medial nucleus of CeA (LeDoux, 2000) (abbreviated as CeC, CeL and CeM, respectively). The LA and BLA mainly contain glutamatergic pyramidal neurons, whereas the CeA is composed of distinct GABAergic neurons. The LA receives multisensory information from the thalamus (LeDoux et al., 1990; Tully et al., 2007), integrated sensory information from the cortex (McDonald, 1998), and noxious stimulus information from the brainstem regions (Johansen et al., 2011). Information flows from the LA and BLA into the CeA, which serves as the amygdala efferents (Duvarci & Pare, 2014). The amygdala is one of the important targets of tachykinins, as demonstrated that the amygdala contains SP (Cassell & Gray, 1989; Shigematsu et al., 2008; Singewald et al., 2008), NKA (Marcos et al., 1998) and NKB (Lucas et al., 1992; Andero et al., 2014; McCullough et al., 2018). Furthermore, the amygdala expresses NK1Rs (Dam et al., 1990b; Sreepathi & Ferraguti, 2012), NK2Rs (Beaujouan et al., 2000; Nagano et al., 2011) and NK3Rs (Dam et al., 1990a; Stoessl & Hill, 1990; Yip & Chahl, 1997; Mileusnic et al., 1999; Nagano et al., 2006; Varnas et al., 2016). With regard to NK3Rs, high densities of NK3R protein (Dam et al., 1990a; Stoessl & Hill, 1990; Mileusnic et al., 1999; Yip & Chahl, 2001) and NKR3 mRNA (Nagano et al., 2006) are distributed in the BLA in animals and in humans (Varnas et al., 2016). While NKB and NK3Rs in the CeA have been reported to be involved in the consolidation of fear memories (Andero et al., 2014; Andero et al., 2016), the functions of NK3Rs in the BLA have not been determined. In the present study, we showed that activation of NK3Rs in the BLA facilitated the excitability of BLA principal neurons by activating TRPC4/5 channels and depressing inwardly rectifying K⁺ (Kir) channels via PLCβ-mediated depletion of PIP₂. We further showed that microinjection of senktide into the BLA increased fear-potentiated startle (FPS) response via activation of TRPC4/5 channels and depression of Kir channels. Our results provide a cellular and molecular mechanism to explain the roles of tachykinins in fear and anxiety responses.

Materials and Methods

Ethical approval

All procedures and experiments presented in this study were approved by the Institutional Animal Use and Care Committee (IACUC) of the University of North Dakota and performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health, USA. Institutional Approval Number 2003-5C.

All the experiments of the present study also comply with the policy and regulations on animal experimentation of The Journal of Physiology (Grundy, 2015).

Preparation of amygdala slices

Coronal brain slices (300 μm) were prepared from virgin male and female Sprague-Dawley (SD) rats (30–45 days old, purchased from Envigo RMS, INC., Indianapolis, IN), knockout (KO) and wild-type (WT) mice (purchased from The Jackson Laboratory). The following three strains of KO mice (1–2 months) and their corresponding age-matched WT mice were used: TRPV1 KO mice (B6.129 × 1-Trpv1^tm1Jul/J, strain 003770) vs. WT mice (C57BL/6J, strain 000664); TRPC4 KO mice (129S1/SvlmJ-Trpc4^tm1.1clph/J, strain 030802) vs. WT mice (129S1/SvlmJ, strain 002448); TRPC5 KO mice (129S1/SvlmJ-Trpc5^tm1.1clph/J, strain 030804) vs. WT mice (129S1/SvlmJ, strain 002448). The animals were housed in the Center for Biomedical Research in the University of North Dakota with food and water available ad libitum. The animal rooms were maintained on a 14/10 h light–dark cycle (lights on at 7:00 a.m.), with a room temperature of 22°C. After being deeply anaesthetized with isoflurane, animals were decapitated and their brains were dissected out. The cerebellum was trimmed and the caudal pole of the brain was glued to the plate of a vibrotome (Leica VT1200S). The cutting solution contained (in mM) 250 sucrose, 104 NaCl, 19.2 NaHCO₃, 2.8 KCl, 1 NaH₂PO₄, 2 CaCl₂, 2.4 MgCl₂ and 8 glucose (saturated with 95% O₂ and 5% CO₂). Cuttings were made from the rostral pole of the brain and slices were collected from both hemispheres when the structure of amygdala appeared. Slices were incubated at 35°C for 30 min in the extracellular solution containing (mM) 60 sucrose, 124 NaCl, 23 NaHCO₃, 3.3 KCl, 1.2 NaH₂PO₄, 2.4 CaCl₂, 1.7 MgCl₂ and 9.5 glucose (saturated with 95% O₂ and 5% CO₂) and then kept at room temperature until use. All animal procedures conformed to the guidelines approved by the University of North Dakota Animal Care and Use Committee.

Electrophysiology

Whole-cell patch-clamp recordings using a Multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) in current- or voltage-clamp mode were made from the principal neurons in the BLA visually identified with infrared video microscopy (Olympus BX51WI) and differential interference contrast optics. The bath was maintained between 33°C and 34°C by an in-line heater and an automatic temperature controller (TC-324C, Warner Instruments). The extracellular solution contained (mM) 130 NaCl, 24 NaHCO₃, 3.5 KCl, 1.25 NaH₂PO₄, 2.5 CaCl₂, 1.5 MgCl₂ and 10 glucose, saturated with 95% O₂ and 5% CO₂. Kynurenic acid (1 mM) and picrotoxin (100 μM) were supplemented in the extracellular solution to block potential indirect actions from synaptic transmission. The recording electrodes were filled with (in mM) 120 K⁺-gluconate, 10 KCl, 5 NaCl, 2 MgCl₂, 10 HEPES, 0.5 EGTA, 2 ATPNa₂, 0.4 GTPNa, and 5 phosphocreatine (pH 7.3). When the Cs⁺-containing intracellular solution was used for recordings, the intracellular K⁺-gluconate was replaced by the same concentration of Cs⁺-gluconate. Because the BLA principal neurons did not show spontaneous action potential (AP) firing, a constant positive current was injected to bring the membrane potential close to the threshold to induce sparse AP firing. The selective NK3R agonist, senktide, was dissolved in the extracellular solution and applied to the cells. To avoid potential desensitization induced by repeated applications of drugs, one slice was limited to only one application of senktide. Data were filtered at 2 kHz, digitized at 10 kHz, acquired online and subsequently analyzed using pCLAMP 10.7 software (Molecular Devices, Sunnyvale, CA). APs were detected in Clampfit 10.7 with ‘Event Detection’ and ‘Threshold Search’. The numbers of APs were binned per minute in Excel. For a subset of experiments, we injected a series of positive currents from 50 pA to 700 pA at an increment of 50 pA every 6 s. This protocol was applied to the same cells before and during the application of senktide for 3–5 min when the maximal effect of senktide was observed.

For the recordings of resting membrane potentials (RMPs), holding currents and current-voltage (I-V) relationship, the extracellular solution was supplemented with tetrodotoxin (TTX, 0.5 μM) to block AP firing. The recording electrodes were filled with the abovementioned K⁺-gluconate-containing intracellular solution. Holding currents were recorded at −60 mV, a membrane potential close to the RMPs of BLA neurons. For the I-V relationship, cells were held at −60 mV and stepped from −140 mV to −40 mV or to +20 mV for 400 ms at a voltage increment of 10 mV every 10 s. Steady-state currents were measured within 5 ms prior to the end of the step voltage protocols. Pharmacological inhibitors were applied to the cells either extracellularly or intracellularly via the recording pipettes. For extracellular application, slices were pretreated for at least 2 h to ensure permeation of reagents into the cells in the slices and the extracellular solution continuously contained the same concentration of the reagents, unless stated otherwise. For intracellular application, we waited for >15 min after the formation of whole‐cell configuration to ensure the diffusion of the inhibitors into the cells.

Surgery, microinjection, and histology

All the procedures for surgery and cannulation were referred from our previous publications (Deng et al., 2009; Xiao et al., 2012; Xiao et al., 2014) and performed under aseptic conditions. Male (195.7 ± 8.4 g, n = 21) and female (168.8 ± 3.9 g, n = 26) SD rats were deeply anaesthetized with 5% vaporized isoflurane and anesthesia was maintained at 3% while rats were placed in a stereotaxic frame (Stoelting Co., IL). Rats were bilaterally implanted with guide cannula (23 GA, 8.5 mm length; P1 Technologies Inc., Roanoke, VA) targeting the BLA with coordinates obtained from the rat brain atlas (from bregma, anteroposterior: −2 ~ −2.6 mm, mediolateral: ± 4.9 ~ 5.0 mm, dorsoventral: −7.2 mm) (Paxinos, 2007). The coordinates were slightly adjusted by the weights of the rats and personal experiences. The end of the guide cannula was positioned 0.5 – 1.0 mm above the BLA. Cannulae were secured in place with dental acrylic bonded to two stainless steel screws (4.8 mm, P1 Technologies Inc., Roanoke, VA) inserted into the skull. Dummy cannulae were screwed into the guide cannulae until microinjection to prevent occlusion. Following surgery, animals were allowed 7–10 days for recovery. Within this recovery period, animals received daily handling for habituation of microinjection procedures. Saline, senktide, SB218795, M084, or tertiapin-Q (all in volume 1 μl per side) were bilaterally injected into the BLA through an internal cannula (30 GA, 8.5 mm; P1 Technologies Inc., Roanoke, VA). Hamilton syringes connected to an automated pump (Harvard Apparatus, MA) via polyethylene tubing were used to administer drug injections at a rate of 1 μl per min. After the injection was completed, the internal cannula was left in place for an additional 2 minutes to ensure adequate diffusion. Following experiments, rats were anesthetized and bilaterally injected with 3% Chicago Sky blue 6B (Sigma) into the BLA (1 μl). Coronal sections were cut on a vibrating microtome (VT1200, Leica Biosystems Inc., IL) and cannula placement was verified. Animals with incorrect cannula placement were excluded from analysis.

Acoustic startle apparatus

Two identical SR-LAB startle chambers with cylindrical animal holders (San Diego Instruments, CA) were used to conduct the FPS experiments. A high-frequency loudspeaker in each startle chamber was mounted 24 cm above the enclosure to elicit white-noise bursts (WNBs). A visual conditioned stimulus was produced by a single lightbulb located in the ceiling of the startle chambers. Foot shocks were used as an unconditioned stimulus and were administered via a stainless-steel floor grid placed in each animal enclosure. The SR-LAB software was used to design and implement the experimental protocols to the rats.

FPS

The paradigm was shown in Figure 6A. On day 1, animals were moved from their home room to the behavioral experiment room for 1 hour. Male and female rats were then placed inside the animal enclosures within the startle chambers for 30 minutes of habituation. On day 2, animals were placed in the startle chamber for a 5-minute habituation period and then presented with 30 WNBs of 95 dB intensity and 50 ms duration at a 30 s intertrial interval (ITI) for startle habituation. The whole session occurred in the presence of a background white-noise (70 dB). On day 3, animals underwent fear conditioning. Animals were microinjected with test compounds 5 minutes prior to fear conditioning, unless otherwise stated. As on days 1 and 2, animals were placed within the enclosures of the startle chambers for a 5-minute acclimation period. Animals were then presented with ten 3.7 s light cues that co-terminate with a 0.5 s foot shock (0.5 mA, pseudorandom ITI 30–180 s). For fear conditioning trials, there was no background white-noise present. On day 4, rats were tested for fear acquisition using a FPS protocol. Following a habituation for 5 minutes inside the startle chambers, the animals were presented with 30 WNB (as above). The 70-dB white noise background was present throughout this trial. The startle amplitude was defined as the maximum peak voltage recorded within the initial 200 ms of the response elicited by the WNBs. The basal amplitudes were the startle amplitudes elicited by the first 10 WNBs in the presence of white noise background alone without the light cue. The remaining 20 WNBs were divided such that 10 WNBs were paired with the light cue (cued) and 10 occurred without (non-cued) in a pseudorandom order. As reported previously (Missig et al., 2010; Moaddab & Dabrowska, 2017), the cued fear responses were expressed as [(amplitude with the light cue - non-cued amplitude)/ non-cued amplitude] × 100%. The non-cued fear responses showed as [(amplitude without the light cue – basal amplitude)/basal amplitude] × 100%. Shock responses recorded during fear conditioning were the maximum peak voltage that occurred during the 0.5 mA shock.

Figure 6: Microinjection of senktide into the BLA dose-dependently enhances cued startle responses.

A, schematic representation of the experimental paradigm. Day 1, animals were habituated within the startle chambers for 30 minutes. Day 2, animals were allowed a 5-minute acclimation and presented with 30 WNBs (95 dB, 50 ms, 30 ITI) in the presence of a background white-noise (70 dB). Day 3, animals were injected with test compounds 5 minutes prior to fear conditioning. Upon placement in the startle chamber, animals were allowed a 5-minute acclimation before presentation of ten 3.7 s light cues that co-terminate with a foot shock (0. 5s, 0.5 mA, pseudorandom ITI 30–180 s). Day 4, rats were allowed a 5-minute acclimation within the startle chamber prior to presentation of 30 WNBs (as on Day 2). The first ten WNBs were used for measurement of basal startle amplitude. The remaining 20 WNBs were divided such that 10 WNBs were paired with light (cued) and 10 occurred without (non-cued) in a pseudorandom order. Background noise was present during this session. B, (left) proper cannula placement and the diffusion area of reagents were verified with bilateral injection of 1 % Chicago Sky Blue dye into the BLA (white arrows); (right) cannula tip placements displayed onto atlas figures adapted from Paxinos and Watson (Paxinos, 2007). Note: all animal cannula placements were verified, dots represent a subset of the rats used for the experiments. C₁, microinjection of senktide dose-dependently increased cued startle responses. C₂, BLA microinjection of senktide had no effect on non-cued startle responses. C₃, startle amplitude was not significantly affected by senktide microinjection. C₄, behavioral responses to foot shock on Day 3 did not differ significantly in response to increasing doses of senktide.

Data analysis and presentation

Data are presented as the means ± SD. Wilcoxon’s matched-pair signed rank test (abbreviated as Wilcoxon test in the text), the Mann–Whitney test, one-way or two-way ANOVA were used for statistical analysis as appropriate. To minimize potential influences of variations from individual animals, each experiment was performed from slices attained from at least four animals and one-way ANOVA was performed to ensure there was no significant difference for the data obtained from individual animals under the same treatment. One-way ANOVA followed by Dunnett’s or Tukey’s multiple comparison test was used for statistical analysis when the pooled control data were used for comparison. Two-way repeated measures ANOVA followed by Sidak’s multiple comparison test was used for statistical analysis for the AP firing frequency elicited by injections of positive currents and for the data used to construct the I-V relationship. Statistical analyses were performed using GraphPad Prism version 9.3 (GraphPad Software Inc., San Diego, CA). P values were reported throughout the text and significance was set as P < 0.05.

Chemicals

The following chemicals were products of R&D Systems: senktide, TTX, picrotoxin, SB218795, ML133, glibenclamide, tertiapin-Q, ML297, M084, U73122, heparin, thapsigargin, chelerythrine and bisindolylmaleimide II (Bis II). Dioctanoyl phosphatidylinositol 4,5-bisphosphate (dic8-PIP₂) was purchased from Echelon Biosciences. The other chemicals were the products of Sigma Aldrich. Drugs were initially prepared in stock solution, aliquoted and stored at −20°C. For those chemicals requiring dimethyl sulfoxide (DMSO) as a solvent, the concentration of DMSO was less than 0.1%.

Results

Activation of NK3Rs excites BLA principal neurons

Because high densities of NK3Rs are expressed in the BLA neurons of a variety of species including the rats (Dam et al., 1990a; Stoessl & Hill, 1990; Mileusnic et al., 1999) and the humans (Mileusnic et al., 1999; Varnas et al., 2016) and activation of NK3Rs increases the expression of c-fos, a marker of neuronal activity, in the amygdala (Yip & Chahl, 1997; Smith & Flynn, 2000), we probed the effects of NK3R activation on the excitability of principal neurons in rat BLA (Fig. 1A). The extracellular solution contained kynurenic acid (1 mM) to block glutamatergic transmission and picrotoxin (100 μM) to block GABAergic transmission. The intracellular solution was the K⁺-gluconate intracellular solution. Under these circumstances, the effects of NK3R agonists should be from the recorded neurons. Bath application of the selective NK3R agonist, senktide at 0.3 μM which is a near-saturating concentration (see below), induced subthreshold depolarization (Control: −63.8 ± 3.1 mV, Senktide: −61.1 ± 3.2 mV, n = 20, P < 0.0001, Wilcoxon test, Fig. 1Ba–b). We then injected a depolarizing current to elevate the membrane potential to just above the firing threshold (−50.1 ± 4.3 mV, n = 16) to elicit sparse AP firing and further tested the effect of senktide on the excitability of BLA neurons. Bath application of the same concentration of senktide for 5 min significantly augmented the AP firing frequency (Control: 0.33 ± 0.23 Hz, Senktide for : 3.71 ± 2.34 Hz, n = 16, P < 0.0001, Wilcoxon test, Fig. 1Ca–c) and the persistent increases of AP firing were still observed after wash in senktide-free extracellular solution for ~1 h (1.80 ± 1.39 Hz, n = 16, P < 0.0001 vs. Control, Wilcoxon test, Fig. 1Ca–c). Furthermore, application of senktide significantly increased AP firing numbers elicited by injecting a series of positive currents from 50 pA to 700 pA at an increment of 50 pA (n = 14, F_(1,13) = 21.26, P = 0.0005, Two-way repeated measures ANOVA followed by Sidak multiple comparison tests, Fig. 1Da–b). These results together indicate that bath application of senktide significantly enhanced the excitability of BLA neurons.

Figure 1. Activation of NK3Rs excites BLA principal neurons.

A, microscopic photograph of amygdala to show the location of BLA where electrophysiological recordings were conducted. LA, lateral nucleus; BLA, basolateral nucleus; CeC, capsular central amygdala; CeL, lateral central amygdala; CeM, medial central amygdala. Ba-Bb, Bath application of the selective NK3R agonist, senktide (0.3 μM), induced subthreshold depolarization of BLA neurons. Ba, RMP recorded from a BLA principal neuron prior to, during and after application of senktide. Bb, Summary data for senktide-elicited subthreshold depolarization. Empty symbols were data from individual cells and solid symbols were their averages (n = 20). Ca-Cc, Application of senktide increased AP firing frequency when a positive current was injected persistently to induce initial sparse AP firing. Ca, APs recorded from a BLA neuron prior to, during and after application of senktide when a positive current was injected persistently to induce basal sparse AP firing. Cb, APs recorded at the time points indicated in Ca in an expanded scale. Cc, Summary data showing senktide-induced excitation of BLA neurons (n = 16). Da-Db, Bath application of senktide augmented AP firing elicited by injection of a series of positive currents from 50 pA to 700 pA at an increment of 50 pA and duration of 600 ms every 6 seconds. Da, APs recorded from a BLA neuron evoked by the positive current injection protocol before (left) and during (right) the application of senktide. Db, Relationship between the injected currents and the elicited AP numbers from 14 BLA neurons. Ea-Eb, Senktide-elicited excitation of BLA neurons was mediated by activation of NK3Rs. Ea, APs recorded from a BLA neuron evoked by the positive current injection protocol before (left) and during (right) the application of senktide in a slice pretreated with the selective NK3 receptor antagonist, SB 218795 (3 μM). The extracellular solution continuously contained the same concentration of SB 218795. Eb, Relationship between the injected currents and the elicited AP numbers from 18 BLA neurons prior to and during the application of senktide in the continuous presence of SB 218795. F, Bath application of neurokinin B (NKB, 0.3 μM), the endogenous NK3 receptor agonist, depolarized BLA neurons. Empty symbols were data from individual cells and solid symbols were their averages (n = 7). Ga-Gc, Application of NKB (0.3 μM) increased AP firing frequency when a positive current was injected persistently to induce initial sparse AP firing. Ga, APs recorded from a BLA neuron prior to, during and after application of NKB when a positive current was injected persistently to induce basal sparse AP firing. Gb, APs recorded at the time points indicated in Ga in an expanded scale. Gc, Summary data showing NKB-induced excitation of BLA neurons (n = 8). Ha-Hb, Application of NKB (0.3 μM) augmented AP firing numbers evoked by injection of a series of positive currents from 50 pA to 700 pA at an increment of 50 pA recorded from the BLA neurons. Ha, APs recorded by the current injection protocol before (left) and during (right) the application of NKB. Hb, Relationship between the injected currents and the elicited AP numbers from 13 BLA neurons before and during the application of NKB.

To test the involvement of NK3Rs, we pretreated the slices with the selective NK3R antagonist, SB 218795 (3 μM) (Giardina et al., 1997) and the bath was continuously perfused with the same concentration of SB 218795. Under these circumstances, bath application of senktide failed to increase AP firing numbers significantly (n = 18, F_(1,17) = 1.609, P = 0.222, Two-way repeated measures ANOVA followed by Sidak multiple comparison tests, Fig. 1Ea–b), suggesting the involvement of NK3Rs. Because neurokinin B (NKB) is an endogenous agonist for NK3Rs, we also tested the effects of NKB on membrane potentials and AP firing numbers. Bath application of NKB (0.3 μM) also elicited subthreshold depolarization (Control: −65.3 ± 4.7 mV, NKB: −61.9 ± 4.6 mV, n = 7, P = 0.016, Wilcoxon test, Fig. 1F). We then injected a persistent positive current to elevate the membrane potentials to just above the firing threshold (−49.9 ± 2.9 mV, n = 8) and tested the effect of NKB on AP firing. Bath application of NKB (0.3 μM) significantly enhanced the AP firing frequency (Control: 0.35 ± 0.25 Hz, NKB: 2.99 ± 1.65 Hz, n = 8, P = 0.008, Wilcoxon test, Fig. 1Ga–c). Likewise, bath application of NKB at the same concentration significantly augmented the firing number of APs evoked by injecting the positive currents from 50 pA to 700 pA at an increment of 50 pA (n = 13, F_(1,12) = 30.91, P = 0.0001, Two-way repeated measures ANOVA followed by Sidak multiple comparison tests, Fig. 1Ha–b). These results together indicate that activation of NK3Rs excites the BLA principal neurons.

Stimulation of NK3Rs activates a cation channel and inhibits an inwardly rectifying K⁺ channel

We further identified the ionic mechanisms whereby activation of NK3Rs facilitates the excitability of BLA neurons. Opening of cation channels and depression of K⁺ channels are the two common ionic mechanisms underlying neuronal excitability. Bath application of senktide (0.3 μM) elicited an inward current recorded at −60 mV with the K⁺-gluconate internal solution (−42.7 ± 20.0 pA, n = 17, P < 0.0001 vs. baseline, Wilcoxon test, Fig. 2Aa and Ad). The EC₅₀ value of senktide was calculated to be 0.064 μM (Fig. 2Ab). We therefore used 0.3 μM senktide for the remaining experiments because this is a near-saturating concentration. Replacement of the K⁺-gluconate-containing intracellular solution with the Cs⁺-gluconate-containing solution to limit the contribution of K⁺ channels significantly (P < 0.0001, Mann-Whitney test) reduced the senktide-induced inward holding currents (−16.7 ± 18.1 pA, n = 25, P < 0.0001 vs. baseline, Wilcoxon test; P < 0.0001 vs. the senktide-induced inward currents recorded in K⁺-containing internal solution, Mann-Whitney test, Fig. 2Ac–d), suggesting that depression of K⁺ channels is one mechanism responsible for NK3R-mediated excitation of BLA neurons.

Figure 2. Senktide-elicited excitation of BLA neurons is mediated by opening a cation channel and inhibiting a Kir channel.

Aa-Ad, Activation of NK3Rs concentration-dependently excited BLA neurons. Aa, Trace of inward current induced by bath application of senktide recorded from a BLA neuron with K⁺-gluconate-containing intracellular solution. Ab, Concentration-response curve of senktide-induced inward currents recorded with K⁺-gluconate-containing intracellular solution. The empty symbols were the inward currents from individual cells and the solid symbols were their averages. Ac, Trace of inward current induced by bath application of senktide recorded from a BLA neuron with Cs⁺-gluconate-containing intracellular solution. Ad, Summary data showing senktide-induced inward currents with K⁺-containing intracellular solution (solid circles) and Cs⁺-containing intracellular solution (empty circles). The bars were their averages. Ba-Bc, Senktide opened a cation channel. The extracellular solution contained TTX (0.5 μM), kynurenic acid (1 mM) and picrotoxin (100 μM) and the intracellular solution was the K⁺-gluconate-containing internal solution. Ba, Currents elicited by a voltage-step protocol before (left) and during (middle) bath application of senktide and the net current obtained by subtraction (right) from a BLA neuron. The dash line was the zero current level. Bb, I-V curve averaged from 11 cells before and during application of senktide (Two-way repeated measures ANOVA followed by Sidak multiple comparison test; Drug: F_{(1, 10)} = 30.03, P = 0.0003; Voltage: F_{(10, 100)} = 42.98, P < 0.0001; Drug × Voltage: F_{(10, 100)} = 3.056, P = 0.002; ** P < 0.0001). Bc, I-V curve of the net current obtained by subtracting the currents in control condition from those after application of senktide. Ca-Cc, Senktide depressed a Kir channel. The extracellular solution contained TTX (0.5 μM), kynurenic acid (1 mM) and picrotoxin (100 μM) and the intracellular solution was the K⁺-gluconate-containing internal solution. Ca, Currents elicited by the voltage-step protocol before (left) and during (middle) bath application of senktide and the net current obtained by subtraction (right) from a BLA neuron. The dash line was the zero current level. Cb, I-V curve averaged from 11 cells before and during application of senktide (Two-way repeated measures ANOVA followed by Sidak multiple comparison test; Drug: F_{(1, 10)} = 2.553, P = 0.141; Voltage: F_{(10, 100)} = 80.87, P < 0.0001; Drug × Voltage: F_{(10, 100)} = 14.51, P < 0.0001; ** P < 0.001). Cc, I-V curve of the net current obtained by subtracting the currents in control condition from those during application of senktide. Da-Dc, Senktide opened a cation channel of outward rectification recorded in the extracellular solution containing 0.5 μM TTX, 1 mM kynurenic acid, 100 μM picrotoxin, 200 μM CdCl₂ and 400 μM NiCl₂ and intracellular solution was Cs⁺-gluconate-containing intracellular solution. Da, Currents elicited by the voltage-step protocol before (left) and during (middle) bath application of senktide and the net current obtained by subtraction (right) from a BLA neuron. The dash line was the zero current level. Db, I-V curve averaged from 18 cells before and during the application of senktide (Two-way repeated measures ANOVA followed by Sidak multiple comparison test; Drug: F_{(1, 17)} = 2.701, P = 0.119; Voltage: F_{(14, 238)} = 146.7, P < 0.0001; Drug × Voltage: F_{(14, 238)} = 27.83, P < 0.0001; * P = 0.05, ** P < 0.01). Dc, I-V curve of the net current obtained by subtracting the currents in control condition from those during application of senktide.

We further measured the I-V relationship of senktide-induced currents to probe the ionic mechanisms whereby activation of NK3Rs excites BLA neurons. Among the 22 BLA pyramidal neurons recorded, 11 cells showed an I-V curve suggestive of non-selective cation channels, i.e., senktide elicited persistent inward currents at the voltages tested (Fig. 2Ba–Bc) and 11 cells displayed an I-V curve resembling that of the inwardly rectifying K⁺ (Kir) channels with a reversal potential at −88.7 ± 17.5 mV (n = 11) (Fig. 2Ca–Cc), close to the calculated K⁺ reversal potential (−95.8 mV). One plausible explanation for the difference of the measured reversal potential and the calculated K⁺ reversal potential is that the latent effect of senktide on the cation channels right-shifted the measured K⁺ reversal potential. We further used Cs⁺-gluconate-containing intracellular solution to block K⁺ channels and included TTX (0.5 μM) to block Na⁺ channels and CdCl₂ (200 μM) and NiCl₂ (400 μM) to block Ca²⁺ channels. We measured the reversal potential of senktide-elicited cation channel currents by extending the voltage ranges to +20 mV. Under these circumstances, the I-V curve of the senktide-induced currents showed outward rectification with a reversal potential of −26.8 ± 18.0 mV (n = 18, Fig. 2Da–Dc). These results together suggest that activation of NK3Rs excites BLA principal neurons by opening a non-selective cation channel and depressing a Kir channel.

We further tested the roles of Kir channels in senktide-induced inward holding currents. Kir channels are sensitive to micromolar concentration of Ba²⁺ (Lacey et al., 1988; Hu et al., 2017; Li et al., 2019). Bath application of Ba²⁺ (500 μM), by itself induced an inward holding current (−42.7 ± 20.4 pA, n = 20, P < 0.0001, Wilcoxon test, Fig. 3A, 3F) and significantly reduced senktide-elicited inward currents (−18.8 ± 13.0 pA, n = 20, P = 0.0008 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 3A, 3G). Kir channels include Kir2, Kir3 (GIRK) and Kir6 (ATP-sensitive, K_ATP) subfamilies and the K⁺ transport channels (Hibino et al., 2010). We utilized ML 133, a specific antagonist for Kir2 subfamily (Wang et al., 2011; Kim et al., 2015; Ford & Baccei, 2016; Sonkusare et al., 2016; Huang et al., 2018) to test the roles of the Kir2 subfamily in senktide-elicited excitation of BLA principal neurons. Bath application of ML 133 (30 μM) by itself did not change the holding currents significantly (−0.02 ± 7.63 pA, n = 15, P = 0.847, Wilcoxon test, Fig. 3B, 3F). In the presence of ML 133, application of senktide still elicited a comparable inward current (−40.9 ± 23.9 pA, n = 15, P = 0.996 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 3B, 3G), suggesting that Kir2 subfamily is not the type of Kir channels involved in senktide-elicited excitation. We then tested the roles of K_ATP channels in senktide-induced inward currents. Bath application of the selective K_ATP channel blocker, glibenclamide (100 μM) did not significantly alter the holding currents in BLA neurons (−4.5 ± 13.7 pA, n = 19, P = 0.490, Wilcoxon test, Fig. 3C, 3F) and exerted no effect on senktide-elicited inward currents (−32.1 ± 22.3 pA, n = 19, P = 0.261 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 3C, 3G), suggesting that K_ATP channels are not involved. We then tested the roles of GIRK (Kir3) subfamily in senktide-elicited excitation. Application of the selective GIRK channel blocker, tertiapin-Q (500 nM) induced an inward current by itself (−30.6 ± 18.6 pA, n = 16, P < 0.0001, Wilcoxon test, Fig. 3D, 3F), suggesting the expression of GIRK channels in the BLA principal neurons. In the continuous presence of tertiapin-Q, application of senktide induced a significantly smaller inward current (−16.0 ± 11.4 pA, n = 15, P = 0.0004 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 3D, 3G), suggesting that GIRK channels are involved in senktide-mediated excitation of BLA pyramidal neurons. Consistent with the involvement of GIRK channels, application of ML 297 (10 μM), an activator of GIRK1-containing channels (Kaufmann et al., 2013), induced an outward current (76.3 ± 50.0 pA, n = 7, P = 0.016 vs. baseline, Wilcoxon test, Fig. 3E–F), suggesting that GIRK1 channels are functionally expressed in the BLA principal neurons and they may be involved in senktide-elicited excitation of BLA principal neurons.

Figure 3. GIRK channels are required for NK3R-elicited inward currents in BLA neurons.

A, current trace recorded from a BLA neuron in response to Ba²⁺ (500 μM) alone and Ba²⁺ plus senktide. B, current trace recorded from a BLA neuron in response to ML133 (30 μM) alone and ML133 plus senktide. C, current trace recorded from a BLA neuron in response to bath application of glibenclamide (100 μM) alone and together with senktide. D, current trace recorded from a BLA neuron in response to tertiapin-Q (500 nM) alone and tertiapin-Q plus senktide. E, current trace recorded from a BLA neuron in response to bath application of ML297 (10 μM). F, summary data for the effects of Kir channel modulators on BLA neurons. **** P < 0.0001 vs. baseline, Wilcoxon test. G, Summary graph showing the effects of Kir channel blockers on senktide-mediated inward currents. *** P < 0.001 vs. senktide alone, one-way ANOVA followed by Dunnett’s test.

TRPC4 and TRPC5 channels are involved in NK3R-elicited excitation of BLA principal neurons

We further identified the cation channels involved in NK3R-elicited excitation of BLA principal neurons. The I-V curve of the senktide-sensitive currents resembles that of TRPC4, TRPC5 and TRPV1 (Wu et al., 2010) channels. The BLA principal neurons express TRPV1 (Zschenderlein et al., 2011; Xiao et al., 2016), TRPC4 (Riccio et al., 2014) and TRPC5 (Riccio et al., 2009) channels. We next tested the roles of these channels in NK3R-mediated excitation of BLA principal neurons. Bath application of the selective TRPC4/5 channel blocker, M084 (100 μM) alone, did not alter the holding currents significantly (3.0 ± 9.5 pA, n = 10, P = 0.375, Wilcoxon test, data not shown). Slices were pretreated with M084 (100 μM), and the extracellular solution was perfused continuously with the same concentration of M084. Application of M084 significantly reduced senktide-induced increases in inward currents (M084 + senktide: −25.0 ± 18.8 pA, n = 19 vs. senktide alone: −42.7 ± 20.0 pA, n = 17, P = 0.019, Mann-Whitney test, Fig. 4Aa–c), suggesting the involvement of TRPC4/5 channels. We further tested the roles of TRPC4/5 channels by using the knockout mice for TRPC4 or TRPC5 channels. Application of senktide induced a significantly smaller inward current in slices prepared from the TRPC4 KO mice (−12.7 ± 7.8 pA, n = 22), compared with the corresponding WT mice (−35.5 ± 15.1 pA, n = 17, P < 0.0001 vs. TRPC4 KO mice, Mann-Whitney test, Fig. 4Ba–c), suggesting the participation of TRPC4 channels in NK3R-elicited excitation of BLA neurons. Likewise, application of senktide evoked a significantly smaller inward current in slices cut from TRPC5 KO mice (−8.7 ± 7.8 pA, n = 22), compared with the corresponding WT mice (−34.6 ± 18.4 pA, n = 17, P < 0.0001, Mann-Whitney test, Fig. 4Ca–c), suggesting the involvement of TRPC5 channels. We also used the TRPV1 KO mice. Application of senktide induced an inward current (−17.1 ± 13.2 pA, n = 36) in slices cut from TRPV1 KO mice, which was not significantly different from that obtained from the corresponding WT mice (−23.7 ± 17.2 pA, n = 22, P = 0.213 vs. TRPV1 KO mice, Mann-Whitney test, Fig. 4Da–c), suggesting that TRPV1 channels are not required for NK3-mediated excitation of BLA neurons.

Figure 4. TRPC4 and TRPC5 channels are involved in NK3R-induced inward currents in BLA neurons.

Aa-Ac: pretreatment of slices with and continuous bath application of M084 (100 μM) significantly reduced senktide-induced inward currents. Aa: senktide-elicited inward current recorded from a BLA neuron in control condition. Ab: senktide-induced inward current recorded from a BLA neuron in a slice treated with the TRPC4/5 channels blocker, M084 (100 μM). Ac: summary graph showing the decreased inward currents induced by senktide in the presence of M084. Circles represented the inward current from individual cells and the bars were their averages. Ba-Bc: bath application of senktide induced a significantly smaller inward current in slices cut from TRPC4 KO mice (TRPC4(−/−)), compared with the corresponding WT mice (TRPC4(+/+)). Ba: senktide-evoked inward current recorded from a BLA neuron in a slice cut from a WT mouse. Bb: senktide-induced inward current recorded from a BLA neuron in a slice cut from a TRPC4 KO mouse. Bc: summary graph showing that senktide induced a significantly smaller inward current in TRPC4 KO mice compared with the corresponding WT mice. Circles represented the inward current from individual cells and the bars were their averages. Ca-Cc: bath application of senktide induced a significantly smaller inward current in slices cut from TRPC5 KO mice (TRPC5(−/−)), compared with the corresponding WT mice (TRPC5(+/+)). Ca: senktide-elicited inward current recorded from a BLA neuron in a slice cut from a WT mouse. Cb: senktide-induced inward current recorded from a BLA neuron in a slice cut from a TRPC5 KO mouse. Cc: summary graph showing that senktide induced a significantly smaller inward current in TRPC5 KO mice compared with the corresponding WT mice. Circles represented the inward current from individual cells and the bars were their averages. Da-Dc: TRPV1 channels were not necessary for NK3R-mediated excitation of BLA neurons. Da: senktide-elicited inward current recorded from a BLA neuron in a slice cut from a WT mouse. Db: senktide-induced inward current recorded from a BLA neuron in a slice cut from a TRPV1 KO mouse. Dc: summary graph indicating that senktide did not significantly alter senktide-induced inward currents in TRPV1 KO mice compared with the corresponding WT mice. Circles represented the inward current from individual cells and the bars were their averages.

NK3R-mediated excitation of BLA neurons is dependent on PLCβ and PLCβ-mediated depletion of PIP₂

Because activation of NK3Rs activates PLCβ pathway (Khawaja & Rogers, 1996; Steinhoff et al., 2014), we tested the roles of PLCβ in NK3R-induced inward currents. Bath application of the selective PLC inhibitor, U73122 (5 μM) had no acute effects on the holding currents recorded from the BLA neurons (−4.82 ± 6.6 pA, n = 5, P = 0.188, Wilcoxon test, data not shown). Pretreatment of slices with and continuous bath application of U73122 (5 μM), significantly reduced senktide-evoked inward currents (−14.4 ± 8.0 pA, n = 16, P < 0.0001 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 5B, 5E), suggesting the involvement of PLCβ. Activation of PLCβ hydrolyzes PIP₂ to generate IP₃ to increase intracellular Ca²⁺ release and DAG to activate PKC. We further tested the roles of these two second messengers in NK3R-mediated inward currents. Inclusion of the IP₃ receptor blocker, heparin (0.5 mg/ml) in the recording pipettes failed to alter senktide-elicited inward currents significantly (−33.0 ± 19.0 pA, n = 16, P = 0.221 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 5C, 5E), suggesting that IP₃ receptors are not involved in NK3R-induced excitation of BLA neurons. Moreover, intracellular application of the sarcoplasmic ATPase inhibitor, thapsigargin (10 μM), to deplete Ca²⁺ stores, did not alter significantly senktide-mediated inward currents (−34.0 ± 15.7 pA, n = 19, P = 0.270 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 5D, 5E), suggesting that intracellular Ca²⁺ release is not required for NK3R-mediated excitation of BLA principal neurons. We then probed the roles of PKC in NK3R-elicited excitation of BLA principal neurons. Pretreatment of slices with and continuous bath application of the selective PKC inhibitor, bisindolylmaleimide II (Bis II, 2 μM), did not significantly alter senktide-induced inward currents (−39.8 ± 25.4 pA, n =12, P = 0.962 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 5F, 5I). Likewise, administration of another PKC inhibitor, chelerythrine (10 μM), in the same fashion, did not significantly change senktide-elicited inward currents (−40.2 ± 22.4 pA, n = 12, P = 0.975 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 5G, 5I). These data together suggest that the function of PKC is irrelevant to NK3R-medited excitation of BLA neurons.

Figure 5. NK3R-elicited excitation of BLA neurons requires PLCβ-mediated depletion of PIP₂.

A, Inward current trace induced by bath application of senktide in control condition. B, Current trace evoked by senktide recorded from a BLA neuron in a slice treated with U73122 (5 μM). C, Senktide-induced inward current recorded from a BLA neuron in a pipette containing heparin (0.5 mg/ml). D, Current trace elicited by senktide in a BLA neuron intracellularly dialyzed with thapsigargin (10 μM). E, Summary graph. Empty symbols were the senktide-induced net currents from individual cells and the bars were their averages. F, Current trace evoked by senktide recorded from a BLA neuron in a slice pretreated and continuously bath-applied with the selective PKC inhibitor, Bis II (2 μM). G, Senktide-induced current trace recorded from a BLA neuron in a slice pretreated and continuously bath-applied with the PKC inhibitor, chelerythrine (10 μM). H, Current trace elicited by senktide recorded from a BLA neuron dialyzed intracellularly with diC8-PIP₂ (40 μM). I, Summary graph. Empty symbols were the senktide-induced net currents from individual cells and the bars were their averages.

We further tested the roles of PLCβ-mediated depletion of PIP₂ in senktide-mediated inward currents. Inclusion of the diC8-PIP₂ (40 μM) to compensate the depletion of PIP₂ content elicited by PLCβ significantly reduced senktide-elicited inward currents (−12.5 ± 13.0 pA, n = 19, P < 0.0001 vs. senktide alone, one-way ANOVA followed by Dunnett’s test, Fig. 5H, 5I), suggesting that PLCβ-mediated depletion of PIP₂ is responsible for NK3R-mediated excitation of BLA neurons.

NK3R-mediated excitation of BLA neurons augments cued startle responses

Because the BLA is closely associated with the FPS responses (Sananes & Davis, 1992; Davis et al., 1993; Kim et al., 1993; Campeau & Davis, 1995; Klumpers et al., 2015), we tested the roles of NK3R-mediated excitation of BLA principal neurons in FPS responses. The FPS paradigm measures conditioned fear by an increase in the amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previously paired with a shock. This test has been proven to be a valuable tool to study mechanisms involved in the acquisition and expression of conditioned fear in both rats and humans (Davis, 1986; Grillon, 2008). We implanted cannulae into the BLA and microinjected senktide and/or other compounds into the BLA to probe the effects and the underlying mechanisms of NK3R activation on FPS by using the paradigm shown in Figure 6A. Figure 6B showed the locations of the cannula tips for a subset of experiments and the diffusion area of Chicago Sky Blue dye injected at the end of the experiments.

Microinjection of senktide into the BLA of rats dose-dependently increased the cued startle response compared with the rats injected with saline (0.9% NaCl) (0.3 nmol, P = 0.047; 1 nmol, P = 0.008, one-way ANOVA followed by Tukey’s test, Fig. 6C₁), whereas senktide had no significantly effects on the non-cued startle response (P = 0.314, one-way ANOVA followed by Tukey’s test, Fig. 6C₂), the startle amplitude (P = 0.063, one-way ANOVA followed by Tukey’s test, Fig. 6C₃) and the shock reactivity amplitude (P = 0.738, one-way ANOVA followed by Tukey’s test, Fig. 6C₄), suggesting that senktide treatment had no effect on generalized stimuli in this paradigm. For the remaining experiments, we used 0.3 nmol senktide because this is an effective dose.

We tested the involvement of NK3Rs by microinjection of the selective NK3R antagonist, SB218795. Microinjection of SB218795 (20 nmol) by itself prior to fear conditioning had no significant effect on cued responses (saline vs SB218795, P = 0.998; one-way ANOVA followed by Tukey’s test), but blocked the enhancement of cued responses elicited by senktide (Senktide alone vs. SB 218795 + Senktide, P =0.006; SB218795 + Senktide vs. saline, P = 0.984; one-way ANOVA followed by Tukey’s test, Fig. 7A), indicating that NK3Rs are responsible for senktide-elicited augmentation of cued-startle response in the BLA.

Figure 7. Roles of NK3Rs, GIRK and TRPC4/5 channels in senktide-mediated augmentation of cued startle responses.

A, Microinjection of senktide (Senk, 0.3 nmol) into the BLA enhanced cued startle responses, whereas prior administration of the NK3R antagonist, SB218795 (20 nmol), GIRK channel blocker, tertiapin-Q (Tert-Q, 250 pmol) or TRPC4/5 channel blocker, M084 (100 nmol) blocked senktide-induced augmentation of cued response. B, non-cued startle responses of the tested compounds and significant enhancements of non-cued startle in the group of rats injected with both tertiapin-Q and senktide and the group of rats injected with M 084. * P < 0.05 vs. saline (one-way ANOVA followed by Tukey’s test). C, group data for startle amplitude on Day 1. D, group data for shock reactivity during fear conditioning on Day 2.

GIRK channels and TRPC4/5 channels are involved in NK3R-mediated enhancement of startle responses

Our results indicate that activation of NK3Rs facilitated the excitabilities of BLA neurons by depression of GIRK channels and opening of TRPC4 and TRPC5 channels. We further examined the roles of these channels in senktide-mediated augmentation of startle responses. Microinjection of tertiapin-Q at 250 pmol, an effective dose demonstrated previously (Mazarati et al., 2006; Morgan et al., 2020), failed to change significantly either the cued (saline vs. tertiapin-Q, P = 0.991; one-way ANOVA followed by Tukey’s test, Fig. 7A) or the non-cued (saline vs. tertiapin-Q, P = 0.677, one-way ANOVA followed by Tukey’s test, Fig. 7B) responses. Microinjection of senktide following tertiapin-Q did not alter significantly the cued startle response (tertiapin-Q vs. tertiapin-Q + senktide, P = 0.992, one-way ANOVA followed by Tukey’s test, Fig. 7A), whereas significantly enhanced the non-cued response (tertiapin-Q vs. tertiapin-Q + senktide, P = 0.009, one-way ANOVA followed by Tukey’s test, Fig. 7B). These results together suggest that GIRK channels are involved in NK3R-mediated enhancement of startle response.

We then tested the involvement of TRPC4/5 channels in NK3R-elicited augmentation of FPS responses. Microinjection of the TRPC4/5 channel blocker, M084 (100 nmol), into the BLA did not significantly alter the cued startle responses (Saline vs. M084, P = 0.997, one-way ANOVA followed by Tukey’s test, Fig. 7A), but blocked senktide-induced enhancement of cued startle response (Saline vs. M084 + Senktide, P = 0.998, one-way ANOVA followed by Tukey’s test, Fig. 7A). However, microinjection of M084 significantly enhanced the non-cued fear response, compared with the saline-injected group (saline vs. M084, P = 0.028, one-way ANOVA followed by Tukey’s test, Fig. 7B). Administration of senktide following the injection of M084 failed to significantly increase the non-cued response further (M084 vs. M084 + senktide, P = 0.125, one-way ANOVA followed by Tukey’s test, Fig. 7B). These results suggest that TRPC4/5 channels are involved in NK3R-elicited enhancement of startle responses.

There were no significant differences for the startle amplitudes obtained during the acoustic startle response session on Day 2 among the experimental groups (P = 0.297, F_(6,40) = 1.26, one-way ANOVA, Fig. 7C), suggesting that there was no endogenous between-group difference in responses to startle-inducing white-noise bursts. The shock reactivity amplitude was unaffected in any group as the result of the microinjection (P = 0.664, F_{(6, 40)} = 0.68, one-way ANOVA, Fig. 7D). Together these data suggest similar behavioral responses to startle-inducing auditory stimuli among these groups and no between-group differences in nociceptive responses to shock resulting from drug injection. Based on our experimental results, we propose a working mode to explain the cellular and molecular mechanisms underpinning NK3R-mediated excitation of BLA neurons and augmentation of FPS response (Fig. 8).

Figure 8. Working mode illustrating the cellular and molecular mechanisms underpinning NK3R-elicited excitation of BLA principal neurons and augmentation of FPS response.

Activation of NK3Rs by senktide results in activation of Gα_q proteins leading to increases in PLCβ activity. Activation of PLCβ catalyzes the hydrolysis of PIP₂ to generate IP₃ to elevate intracellular Ca²⁺ release from IP₃-sensitive store and diacylglycerol (DAG) to activate protein kinase C (PKC). PLCβ-induced depletion of PIP₂ results in depression of GIRK channels and opening of TRPC4/5 channels to excite BLA principal neurons. The effects of NK3Rs on GIRK channels and TRPC4/5 channels are responsible for NK3R-induced augmentation of FPS response.

Discussion

While high densities of NK3Rs are expressed in the BLA (Dam et al., 1990a; Stoessl & Hill, 1990; Mileusnic et al., 1999; Yip & Chahl, 2001; Nagano et al., 2006), the actions and the underlying mechanisms of NK3R activation in the BLA have not been determined. We showed that activation of NK3Rs excited BLA neurons assessed by electrophysiologically recording AP firing, RMPs and holding currents. Consistent with our electrophysiological results, intracerebroventricular injection of senktide increases the expression of c-Fos, a selective marker of neuronal activity in the BLA (Yip & Chahl, 1997; Smith & Flynn, 2000). Our results further showed that activation of NK3Rs facilitated the excitabilities of BLA neurons by depressing the GIRK type of Kir channels and opening TRPC4 and TRPC5 channels. While SP has been shown to facilitate neuronal excitability by depressing Kir channels (Stanfield et al., 1985; Yamaguchi et al., 1990; Bajic et al., 2002) or by activating cation channels (Aosaki & Kawaguchi, 1996) or by both suppressing Kir conductance and activating cation channels (Shen & North, 1992; Koyano et al., 1993; Drew et al., 2005), few studies have been conducted to determine the ionic mechanisms underlying NK3R-mediated neuronal excitation. Although SP is capable of inhibiting the GIRK type of Kir channels (Bajic et al., 2002; Mao et al., 2004), the subtypes of the Kir channels involved in NK3R-mediated facilitation of neuronal excitability have not been determined. Furthermore, the cation channels underlying tachykinins-mediated neuronal excitation have not been identified. With both pharmacological approaches and KO mice, we identified that GIRK channels and TRPC4 and TRPC5 channels are the type of Kir and cation channels involved in NK3R-induced excitation of BLA neurons. GIRK channels comprise four isoforms, namely GIRK1, GIRK2, GIRK3 and GIRK4. Consistent with our results, amygdala abundantly express GIRK1, GIRK2 and GIRK3 (Karschin et al., 1996; Sosulina et al., 2008). Our result that application of the selective GIRK1 activator, ML 297, elicited an outward current in the BLA neurons, further supports the expression of functional GIRK1 channels in these neurons. GIRK channels exist as predominantly heterotetramers of GIRK1, GIRK2 and/or GIRK3, or as homotetramers of the GIRK2 subunit (Hibino et al., 2010; Luscher & Slesinger, 2010). NK3R-elicited excitation of BLA neurons might be mediated by suppression of the heterotetramers formed by GIRK1, GIRK2 and/or GIRK3, or the homotetramers of the GIRK2 subunit. Future experiments are required to identify the isoform(s) of GIRK channels involved in NK3R-elicited excitation of BLA neurons. Our results further showed that activation of NK3Rs excited about half of the BLA principal neurons by opening TRPC4 and TRPC5 channels. Consistent with our results, both TRPC4 (Riccio et al., 2014) and TRPC5 (Riccio et al., 2009) channels are expressed in the BLA and genetic deletion of these channels decreased fear and anxiety-related responses (Riccio et al., 2009; Riccio et al., 2014).

Our results indicated that PLCβ is required for NK3R-induced inward currents. This result is in line with previous results showing that PLCβ1 is involved in SP-elicited depression of Kir channels in the cholinergic neurons from the nucleus basalis (Takano et al., 1996). Whereas previous studies failed to determine whether intracellular Ca²⁺ release is required for SP-induced depression of Kir channels, our results showed that intracellular Ca²⁺ release from the IP₃ store is unnecessary for NK3R-generated inward currents. However, there is considerable discrepancy for the roles of PKC in NK3R-produced inward currents and SP-induced inhibition of Kir channels. Whereas PKC is involved in SP-mediated inhibition of Kir channels in nucleus basalis neurons (Takano et al., 1995; Nakajima & Nakajima, 2010) and SP-induced inhibition of GIRK1/GIRK4 channels expressed in Xenopus oocytes (Mao et al., 2004), our results failed to detect a role of PKC in NK3R-elicited inward currents in BLA neurons. Several possibilities could be proposed to explain the discrepancy. First, the Kir channels depressed by SP in the nucleus basalis is distinct from the GIRK channels as the single channel conductance depressed by SP (~23 pS) is different from that of GIRK channels (32–35 pS) (Bajic et al., 2002; Nakajima & Nakajima, 2010). Second, there are controversial results as to whether PKC is required for the G_q/11-coupled receptor-mediated depression of GIRK channels. PKC has been shown to phosphorylate and depress GIRK channels (Stevens et al., 1999; Mao et al., 2004; Adney et al., 2015; Niemeyer et al., 2019; Hu et al., 2020). There is also evidence demonstrating that the function of PKC is unnecessary for the depression of GIRK channels induced by G_q/11‐coupled receptors (Mark & Herlitze, 2000; Lei et al., 2003).

We further tested the roles of PIP₂, the upstream signal of PKC, in NK3R-produced inward currents, as PIP₂ has been shown to regulate functionally a variety of ion channels (Suh & Hille, 2008; Rodriguez-Menchaca et al., 2012). Intracellular dialysis of diC8‐PIP₂ significantly reduced senktide-induced inward currents in BLA neurons, suggesting that PLCβ-elicited depletion of PIP₂ is involved in NK3R-mediated excitation of BLA neurons. As our results indicate that both depression of GIRK channels and activation of TRPC4/5 channels are involved in NK3R-induced inward currents, these results also suggest that PLCβ-mediated depletion of PIP₂ is responsible for NK3R-elicited suppression of GIRK channels and activation of TRPC4/5 channels. Consistent with this scenario, PLCβ-mediated PIP₂ depletion is responsible for G_q/11 receptor-elicited depression of GIRK (Mark & Herlitze, 2000; Cho et al., 2001; Meyer et al., 2001; Lei et al., 2003; Cho et al., 2005; Keselman et al., 2007; Whorton & MacKinnon, 2011) and activation of TRPC4 (Otsuguro et al., 2008) and TRPC5 (Trebak et al., 2009; Ningoo et al., 2021) channels. PIP₂ decreases the desensitization of TRPC5 channels and PLCβ-induced hydrolysis of PIP₂ may increase the desensitization of the channels and thus facilitate the closing of the channels (Ningoo et al., 2021).

Fear is a response to impending threat that prepares a subject to make appropriate defensive responses including freezing, fighting, or fleeing to safety. In humans, fear is accompanied by affective feelings of dread and anticipation. The FPS paradigm has been proven to be a useful system with which to analyze neural systems involved in fear and anxiety. This test measures conditioned fear by an increase in the amplitude of a simple reflex (the acoustic startle reflex) in the presence of a cue previously paired with a shock. The hypothesis to explain the FPS is that the conditioned stimulus activates the CeA through a pathway involving the lateral geniculate nucleus, perirhinal cortex, LA and BLA. The CeA then projects directly to the acoustic startle pathway so as to modulate the startle response (Davis, 1993; Davis et al., 1993; Davis, 2006).The BLA may be a neural substrate for the acquisition of conditioned fear responses (Sananes & Davis, 1992; Davis et al., 1993; Kim et al., 1993; Campeau & Davis, 1995; Klumpers et al., 2015). Because the BLA is an important structure in the neural circuitry underlying FPS, NK3R-mediated excitation of BLA principal neurons likely augmented the output of information from the BLA and up-regulated the activity in the circuitry, resulting in elevated FPS response. Furthermore, it has been demonstrated that the functions of NMDA receptors are required for FPS as infusion of the NMDA receptor antagonist APV into the BLA impairs FPS (Miserendino et al., 1990; Campeau et al., 1992; Gewirtz & Davis, 1997; Walker & Davis, 2000). Because NMDA receptors are voltage-dependently blocked by Mg²⁺, the subthreshold depolarization elicited by the activation of NK3Rs could relieve the Mg²⁺-block of NMDA receptors and thus augment FPS. Consistent with our results, intracerebroventricular administration of the selective NK3R agonist, senktide, evokes gerbil foot tapping which is thought to reflect a fear-related response (Sundqvist et al., 2007). Likewise, NK3Rs may be responsible for the anxiogenic-like actions of SP6-11(C-terminal), a specific metabolite of SP (Duarte et al., 2016). More specifically, up-regulation of NKB and NK3Rs in the CeM facilitates the consolidation of fear memories (Andero et al., 2016). All these results together suggest that NKB/NK3R system facilitates fear and anxiety-like responses and antagonists of NK3Rs could be potential therapeutic agents for anxiety treatment.

The results that microinjection of tertiapin-Q with senktide or M084 alone enhanced the non-cued responses (Figure 7B) are unexpected. These results may be explained by the complicated roles of Kir channels and TRPC4/5 channels in anxiety and fear responses. Conflicting results have been obtained as to the roles of GIRK channels in anxiety and fear responses. GIRK1 or GIRK2 knockout mice showed reduced anxiety-related behavior in the elevated plus maze (Pravetoni & Wickman, 2008), although constitutive GIRK2 knockout mice exhibited a striking deficit in hippocampal-dependent (contextual) and hippocampal-independent (cue) fear conditioning (Victoria et al., 2016). However, application of the GIRK1 activator ML297 decreased anxiety-related behavior (Wydeven et al., 2014) and fear conditioning increased the activity of BLA neurons by suppression of K⁺ channels (Sun et al., 2015). The non-cued responses may represent the background anxiety possibly due to the repeated applications of the light cues (Missig et al., 2010; Ayers et al., 2016). Tertiapin-Q-mediated inhibition of GIRK channels might have already altered the anxiety level, which might have synergized with subsequent application of NK3R agonist senktide and thus an increase in non-cued responses was observed. The increasing effect of M084 on non-cued responses could be an off-target actions of M084 because M084 is an acetylcholinesterase inhibitor as well (Zhu et al., 2013). As acetylcholine efflux in the BLA has been observed during fear conditioning session (Kellis et al., 2020), application of M084 could have augmented the non-cued responses by interacting with other neurotransmitters such as acetylcholine. While these results are perplexing, our results in general provide a cellular and molecular mechanism to explain the augmentation of fear response in response to NK3R activation.

Key Points Summary.

• Activation of NK3 receptors (NK3Rs) facilitates the excitability of principal neurons in rat basolateral amygdala (BLA)

• NK3R-induced excitation is mediated by inhibition of GIRK channels and activation of TRPC4/5 channels

• PLCβ and depletion of PIP₂ are necessary for NK3R-mediated excitation of BLA principal neurons

• Activation of NK3Rs in the BLA facilitates fear-potentiated startle (FPS) response

• GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of FPS

Translational Perspective:

Amygdala is an essential structure involved in anxiety and fear responses. Neurokinin B (NKB) is a peptide which plays a neural modulation role in the brain. Because high density of NKB receptors named as NK3 receptors are expressed in the basolateral amygdala (BLA), we tested the hypothesis that activation of NK3 receptors facilitates the excitability of BLA neurons and fear responses. Our results demonstrate that NK3 receptor activity enhances neuronal excitability in the BLA by depressing the G-protein-gated inwardly rectifying potassium (GIRK) channels and activating TRPC4/5 channels. Phospholipase C-mediated depletion of phosphatidylinositol biphosphate (PIP₂) is responsible for NK3 receptor-mediated enhancement of neuronal excitability. We further found that activation of NK3 receptors in the BLA increases fear responses via depression of GIRK channels and activation of TRPC4/5 channels. Our results suggest that antagonism of NK3 receptors could be a potential mechanism to relieve fear and anxiety responses.

Biography

Cody Boyle is a PhD candidate at the University of North Dakota working in the laboratory of Dr. Saobo Lei. His interests include the neurobiology of emotional memory and anxiety-related disorders. Currently, he is focused on the role neuropeptides play on synaptic activity and neuronal circuitry in the amygdala. He received his BS and MS in Biology with Dr. Tristan Darland. His MS focused on the epigenetic and behavioral outcomes resulting from early embryonic cocaine exposure in a zebrafish model.

Data Availability Statement:

The data that support the findings of this study are available from the corresponding author upon reasonable request.