Leptin excites basolateral amygdala principal neurons and reduces food intake by LepRb-JAK2-PI3K-dependent depression of GIRK channels.

Leptin is an adipocyte-derived hormone that modulates food intake, energy balance, neuroendocrine status, thermogenesis, and cognition. Whereas a high density of leptin receptors has been detected in the basolateral amygdala (BLA) neurons, the physiological functions of leptin in the BLA have not been determined yet. We found that application of leptin excited BLA principal neurons by activation of the long form leptin receptor, LepRb. The LepRb-elicited excitation of BLA neurons was mediated by depression of the G protein-activated inwardly rectifying potassium (GIRK) channels. Janus Kinase 2 (JAK2) and phosphoinositide 3-kinase (PI3K) were required for leptin-induced excitation of BLA neurons and depression of GIRK channels. Microinjection of leptin into the BLA reduced food intake via activation of LepRb, JAK2, and PI3K. Our results may provide a cellular and molecular mechanism to explain the physiological roles of leptin in vivo.

Neuromedin B excites central lateral amygdala neurons and reduces cardiovascular output and fear-potentiated startle.

Neuromedin B (NMB) and gastrin-releasing peptide (GRP) are the two mammalian analogs in the bombesin peptide family that exert a variety of actions including emotional processing, appetitive behaviors, cognition, and tumor growth. The bombesin-like peptides interact with three receptors: the NMB-preferring bombesin 1 (BB1) receptors, the GRP-preferring bombesin 2 (BB2) receptors and the orphan bombesin 3 (BB3) receptors. Whereas, injection of bombesin into the central amygdala reduces satiety and modulates blood pressure, the underlying cellular and molecular mechanisms have not been determined. As administration of bombesin induces the expression of Fos in the lateral nucleus of the central amygdala (CeL) which expresses BB1 receptors, we probed the effects of NMB on CeL neurons using in vitro and in vivo approaches. We showed that activation of the BB1 receptors increased action potential firing frequency recorded from CeL neurons via inhibition of the inwardly rectifying K+ (Kir) channels. Activities of phospholipase Cß and protein kinase C were required, whereas intracellular Ca2+ release was unnecessary for BB1 receptor-elicited potentiation of neuronal excitability. Application of NMB directly into the CeA reduced blood pressure and heart rate and significantly reduced fear-potentiated startle. We may provide a cellular and molecular mechanism whereby bombesin-like peptides modulate anxiety and fear responses in the amygdala.

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

The tachykinin peptides include substance P (SP), neurokinin A and neurokinin B, 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 non-selective 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 Cß (PLCß), but were independent of intracellular Ca2+ release and protein kinase C. PLCß-mediated depletion of phosphatidylinositol 4,5-bisphosphate 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. KEY POINTS: 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. Phospholipase Cß and depletion of phosphatidylinositol 4,5-bisphosphate are necessary for NK3R-mediated excitation of BLA principal neurons. Activation of NK3Rs in the BLA facilitates fear-potentiated startle response. GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of fear-potentiated startle.

PLCß-Mediated Depletion of PIP2 and ATP-Sensitive K+ Channels Are Involved in Arginine Vasopressin-Induced Facilitation of Neuronal Excitability and LTP in the Dentate Gyrus.

Arginine vasopressin (AVP) serves as a neuromodulator in the brain. The hippocampus is one of the major targets for AVP, as it has been demonstrated that the hippocampus receives vasopressinergic innervation and expresses AVP receptors. The dentate gyrus (DG) granule cells (GCs) serve as a gate governing the inflow of information to the hippocampus. High densities of AVP receptors are expressed in the DG GCs. However, the roles and the underlying cellular and molecular mechanisms of AVP in the DG GCs have not been determined. We addressed this question by recording from the DG GCs in rat hippocampal slices. Our results showed that application of AVP concentration-dependently evoked an inward holding current recorded from the DG GCs. AVP depolarized the DG GCs and increased their action potential firing frequency. The excitatory effects of AVP were mediated by activation of V1a receptors and required the function of phospholipase Cß (PLCß). Whereas intracellular Ca2+ release and protein kinase C activity were unnecessary, PLCß-induced depletion of phosphatidylinositol 4,5-bisphosphate was involved in AVP-evoked excitation of the DG GCs. AVP excited the DG GCs by depression of the ATP-sensitive K+ channels, which were required for AVP-elicited facilitation of long-term potentiation at the perforant path-GC synapses. Our results may provide a cellular and molecular mechanism to explain the physiological functions of AVP, such as learning and memory, and pathologic disorders like anxiety.

Roles of PLCß, PIP2 , and GIRK channels in arginine vasopressin-elicited excitation of CA1 pyramidal neurons.

Arginine vasopressin (AVP) is a hormone exerting vasoconstrictive and antidiuretic action in the periphery and serves as a neuromodulator in the brain. Although the hippocampus receives vasopressinergic innervation and AVP has been shown to facilitate the excitability of CA1 pyramidal neurons, the involved ionic and signaling mechanisms have not been determined. Here we found that AVP excited CA1 pyramidal neurons by activation of V1a receptors. Functions of G proteins and phospholipase Cß (PLCß) were required for AVP-elicited excitation of CA1 pyramidal neurons, whereas intracellular Ca2+ release and protein kinase C were unnecessary. PLCß-mediated depletion of phosphatidylinositol 4,5-bisphosphate (PIP2 ) was required for AVP-elicited excitation of CA1 pyramidal neurons. AVP augmented the input resistance and increased the time constants of CA1 pyramidal neurons. AVP induced an inward current in K+ -containing intracellular solution, whereas no inward currents were observed with Cs+ -containing intracellular solution. AVP-sensitive currents showed inward rectification with a reversal potential close to the K+ reversal potential, suggesting the involvement of inwardly rectifying K+ channels. AVP-induced currents were sensitive to the micromolar concentration of Ba2+ and tertiapin-Q, whereas application of ML 133, a selective Kir2 channel blocker had no effects, suggesting that AVP excited CA1 pyramidal neurons by depressing G protein-gated inwardly rectifying K+ channels. Activation of V1a receptors in the CA1 region facilitated glutamatergic transmission onto subicular pyramidal neurons, suggesting that AVP modulates network activity in the brain. Our results may provide one of the cellular and molecular mechanisms to explain the in vivo physiological functions of AVP.

Involvement of TRPC5 channels, inwardly rectifying K+ channels, PLCß and PIP2 in vasopressin-mediated excitation of medial central amygdala neurons.

KEY POINTS: Activation of V1a vasopressin receptors facilitates neuronal excitability in the medial nucleus of central amygdala (CeM) V1a receptor activation excites about 80% CeM neurons by opening a cationic conductance and about 20% CeM neurons by suppressing an inwardly rectifying K+ (Kir) channel The cationic conductance activated by V1a receptors is identified as TRPC5 channels PLCß-mediated depletion of PIP2 is involved in V1a receptor-elicited excitation of CeM neurons Intracellular Ca2+ release and PKC are unnecessary for V1a receptor-mediated excitation of CeM neurons ABSTRACT: Arginine vasopressin (AVP) serves as a hormone in the periphery to modulate water homeostasis and a neuromodulator in the brain to regulate a diverse range of functions including anxiety, social behaviour, cognitive activities and nociception. The amygdala is an essential brain region involved in modulating defensive and appetitive behaviours, pain and alcohol use disorders. Whereas activation of V1a receptors in the medial nucleus of the central amygdala (CeM) increases neuronal excitability, the involved ionic and signalling mechanisms have not been determined. We found that activation of V1a receptors in the CeM facilitated neuronal excitability predominantly by opening TRPC5 channels, although AVP excited about one fifth of the CeM neurons via suppressing an inwardly rectifying K+ (Kir) channel. G proteins and phospholipase Cß (PLCß) were required for AVP-elicited excitation of CeM neurons, whereas intracellular Ca2+ release and the activity of protein kinase C were unnecessary. Prevention of the depletion of phosphatidylinositol 4,5-bisphosphate (PIP2 ) blocked AVP-induced excitation of CeM neurons, suggesting that PLCß-mediated depletion of PIP2 is involved in AVP-mediated excitation of CeM neurons. Our results may provide a cellular and molecular mechanism to explain the anxiogenic effects of AVP in the amygdala.

Activation of Oxytocin Receptors Excites Subicular Neurons by Multiple Signaling and Ionic Mechanisms.

Oxytocin (OXT) is a nonapeptide that serves as a neuromodulator in the brain and a hormone participating in parturition and lactation in the periphery. The subiculum is the major output region of the hippocampus and an integral component in the networks that process sensory and motor cues to form a cognitive map encoding spatial, contextual, and emotional information. Whilst the subiculum expresses the highest OXT-binding sites and is the first brain region to be activated by peripheral application of OXT, the precise actions of OXT in the subiculum have not been determined. Our results demonstrate that application of the selective OXT receptor (OXTR) agonist, [Thr4,Gly7]-oxytocin (TGOT), excited subicular neurons via activation of TRPV1 channels, and depression of K+ channels. The OXTR-mediated excitation of subicular neurons required the functions of phospholipase Cß, protein kinase C, and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2). OXTR-elicited excitation of subicular neurons enhanced long-term potentiation via activation of TRPV1 channels. Our results provide a cellular and molecular mechanism to explain the physiological functions of OXT in the brain.

Oxytocin receptors excite lateral nucleus of central amygdala by phospholipase Cß- and protein kinase C-dependent depression of inwardly rectifying K+ channels.

KEY POINTS: Activation of oxytocin receptors (OXTRs) facilitates neuronal excitability in rat lateral nucleus of central amygdala (CeL). OXTR-induced excitation is mediated by inhibition of inwardly rectifying K+ (Kir) channels. Phospholipase Cß is necessary for OXTR-mediated excitation of CeL neurons and depression of Kir channels. OXTR-elicited depression of Kir channels and excitation of CeL neurons require the function of Ca2+ -dependent protein kinase C. ABSTRACT: Oxytocin (OXT) is a nonapeptide that exerts anxiolytic effects in the brain. The amygdala is an important structure involved in the modulation of fear and anxiety. A high density of OXT receptors (OXTRs) has been detected in the capsular (CeC) and lateral (CeL) nucleus of the central amygdala (CeA). Previous studies have demonstrated that activation of OXTRs induces remarkable increases in neuronal excitability in the CeL/C. However, the signalling and ionic mechanisms underlying OXTR-induced facilitation of neuronal excitability have not been determined. We found that activation of OXTRs in the CeL increased action potential firing frequency recorded from neurons in this region via inhibition of the inwardly rectifying K+ channels. The functions of phospholipase Cß and protein kinase C were required for OXTR-induced augmentation of neuronal excitability. Our results provide a cellular and molecular mechanism whereby activation of OXTRs exerts anxiolytic effects.

Roles of K+ and cation channels in ORL-1 receptor-mediated depression of neuronal excitability and epileptic activities in the medial entorhinal cortex.

Nociceptin (NOP) is an endogenous opioid-like peptide that selectively activates the opioid receptor-like (ORL-1) receptors. The entorhinal cortex (EC) is closely related to temporal lobe epilepsy and expresses high densities of ORL-1 receptors. However, the functions of NOP in the EC, especially in modulating the epileptiform activity in the EC, have not been determined. We demonstrated that activation of ORL-1 receptors remarkably inhibited the epileptiform activity in entorhinal slices induced by application of picrotoxin or by deprivation of extracellular Mg2+. NOP-mediated depression of epileptiform activity was independent of synaptic transmission in the EC, but mediated by inhibition of neuronal excitability in the EC. NOP hyperpolarized entorhinal neurons via activation of K+ channels and inhibition of cation channels. Whereas application of Ba2+ at 300 µM which is effective for the inward rectifier K+ (Kir) channels slightly inhibited NOP-induced hyperpolarization, the current-voltage (I-V) curve of the net currents induced by NOP was linear without showing inward rectification. However, a role of NOP-induced inhibition of cation channels was revealed after inhibition of Kir channels by Ba2+. Furthermore, NOP-mediated augmentation of membrane currents was differently affected by application of the blockers selective for distinct subfamilies of Kir channels. Whereas SCH23390 or ML133 blocked NOP-induced augmentation of membrane currents at negative potentials, application of tertiapin-Q exerted no actions on NOP-induced alteration of membrane currents. Our results demonstrated a novel cellular and molecular mechanism whereby activation of ORL-1 receptors depresses epilepsy.

Longitudinal Effects of Embryonic Exposure to Cocaine on Morphology, Cardiovascular Physiology, and Behavior in Zebrafish.

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults.