Rat Locus Coeruleus Neurons Research Articles

Pharmacological characterization of prostaglandin E2 EP2 and EP4 receptors in male rat locus coeruleus neurons ex vivo

The inflammatory mediator prostaglandin E2 (PGE2) binds to Gs-coupled EP2 and EP4 receptors. These receptors are located in the locus coeruleus (LC), the principal noradrenergic nucleus in the brain, but their functional role remains unknown. In this study, the PGE2 EP2 and EP4 receptors in LC cells from male rat brain slices were pharmacologically characterized by single-unit extracellular electrophysiology. The EP2 receptor agonists butaprost (0.01–10 μM) and treprostinil (0.03–10 µM) and the EP4 receptor agonists rivenprost (0.01 nM–1 µM) and TCS2510 (0.20 nM–2 µM) increased the firing rate of LC neurons in a concentration-dependent manner. The EP2 receptor antagonist PF-04418948 (10 nM) hindered the excitatory effect of butaprost and treprostinil while the EP4 receptor antagonist L-161,982 (30 and 300 nM) blocked the excitatory effect caused by rivenprost and TCS2510. The effects of butaprost and rivenprost were prevented by extracellular sodium replacement but were not modified by the protein kinase A (PKA) activator 8-Br-cAMP (1 mM) or the inhibitor H-89 (10 μM). However, the Gβγ subunit blocker gallein (20 μM) hindered the stimulatory effect of butaprost while the Gαs subunit inhibitor NF449 (10 µM) prevented that of rivenprost. Finally, rivenprost-induced stimulation (30 nM) was not occluded by butaprost (1 µM).In conclusion, activation of EP2 and EP4 receptors excites LC noradrenergic neurons through sodium-dependent currents via different G protein subunits in male rat brain slices. EP2 and EP4 in the LC may constitute pharmacological targets for the treatment of pain, fever, drug addiction, anxiety and neuroinflammatory diseases.

Inhibition of rat locus coeruleus neurons by prostaglandin E2 EP3 receptors: pharmacological characterization ex vivo.

Prostaglandin E2 (PGE2) is an inflammatory mediator synthesized by the brain constitutive cyclooxygenase enzyme. PGE2 binds to G protein-coupled EP1-4 receptors (EP1 to Gq, EP2,4 to Gs, and EP3 to Gi/o). EP2, EP3 and EP4 receptors are expressed in the locus coeruleus (LC), the main noradrenergic nucleus in the brain. EP3 receptors have been explored in the central nervous system, although its role regulating the locus coeruleus neuron activity has not been pharmacologically defined. Our aim was to characterize the function of EP3 receptors in neurons of the LC. Thus, we studied the effect of EP3 receptor agonists on the firing activity of LC cells in rat brain slices by single-unit extracellular electrophysiological techniques. The EP3 receptor agonist sulprostone (0.15nM-1.28µM), PGE2 (0.31nM-10.2µM) and the PGE1 analogue misoprostol (0.31nM-2.56µM) inhibited the firing rate of LC neurons in a concentration-dependent manner (EC50 = 15nM, 110nM, and 51nM, respectively). The EP3 receptor antagonist L-798,106 (3-10µM), but not the EP2 (PF-04418948, 3-10µM) or EP4 (L-161,982, 3-10µM) receptor antagonists, caused rightward shifts in the concentration-effect curves for the EP3 receptor agonists. Sulprostone-induced effect was attenuated by the Gi/o protein blocker pertussis toxin (pertussis toxin, 500ngml-1) and the inhibitors of inwardly rectifying potassium channels (GIRK) BaCl2 (300µM) and SCH-23390 (15µM). In conclusion, LC neuron firing activity is regulated by EP3 receptors, presumably by an inhibitory Gi/o protein- and GIRK-mediated mechanism.

Acute morphine injection persistently affects the electrophysiological characteristics of rat locus coeruleus neurons

Administration of morphine is associated with critical complications in clinic which primarily includes the development of dependence and tolerance even following a single dose (acute) exposure. Behavioral and electrophysiological studies support the significant role of locus coeruleus (LC) neurons in tolerance and dependence following chronic morphine exposure. The current study was designed to explore the electrophysiological properties of the LC neurons following acute morphine exposure. In-vitro whole-cell patch-clamp recordings were performed in LC neurons 24 h after intraperitoneal morphine injection. Acute morphine injection significantly decreased the spontaneous firing rate of LC neurons, the rising and decay slopes of action potentials, and consequently increased the action potential duration. In addition, morphine treatment did not alter the rheobase current and first spike latency while affected the inhibitory postsynaptic currents elicited in response to orexin-A. In fact, single morphine exposure could inhibit the disinhibitory effect of orexin-A on LC neurons.

Central blockade of orexin type 1 receptors reduces naloxone induced activation of locus coeruleus neurons in morphine dependent rats

Orexin neuropeptides are implicated in the expression of morphine dependence. Locus coeruleus (LC) nucleus is an important brain area involving in the development of withdrawal signs of morphine and contains high expression of orexin type 1 receptors (OX1Rs). Despite extensive considerations, effects of immediate inhibition of OX1Rs by a single dose administration of SB-334867 prior to the naloxone-induced activation of LC neurons remains unknown. Therefore, we examined the direct effects of OX1Rs acute blockade on the neuronal activity of the morphine-dependent rats which underwent naloxone administration. Adult male rats underwent subcutaneous administration of 10 mg/kg morphine (two times/day) for a ten-day period. On the last day of experiment, intra-cerebroventricular administration of 10 μg/μl antagonist of OX1Rs, SB-334867, was performed just before intra-peritoneal injection of 2 mg/kg naloxone. Thereafter, in vivo extracellular single unit recording was employed to evaluate the electrical activity of LC neuronal cells. The outcomes demonstrated that morphine tolerance developed following ten-day of injection. Then, naloxone administration causes hyperactivity of LC neuronal cells, whereas a single dose administration of SB-334867 prior to naloxone prevented the enhanced activity of neurons upon morphine withdrawal. Our findings indicate that increased response of LC neuronal cells to applied naloxone could be prevented by the acute inhibition of the OX1Rs just before the naloxone treatment.

Locus Coeruleus Activation Patterns Differentially Modulate Odor Discrimination Learning and Odor Valence in Rats.

ABSTRACTThe locus coeruleus (LC) produces phasic and tonic firing patterns that are theorized to have distinct functional consequences. However, how different firing modes affect learning and valence encoding of sensory information are unknown. Here, we show bilateral optogenetic activation of rat LC neurons using 10-Hz phasic trains of either 300 ms or 10 s accelerated acquisition of a similar odor discrimination. Similar odor discrimination learning was impaired by noradrenergic blockade in the piriform cortex (PC). However, 10-Hz phasic light-mediated learning facilitation was prevented by a dopaminergic antagonist in the PC, or by ventral tegmental area (VTA) silencing with lidocaine, suggesting a LC–VTA–PC dopamine circuitry involvement. Ten-hertz tonic stimulation did not alter odor discrimination acquisition, and was ineffective in activating VTA DA neurons. For valence encoding, tonic stimulation at 25 Hz induced conditioned odor aversion, whereas 10-Hz phasic stimulations produced an odor preference. Both conditionings were prevented by noradrenergic blockade in the basolateral amygdala (BLA). Cholera Toxin B retro-labeling showed larger engagement of nucleus accumbens-projecting neurons in the BLA with 10-Hz phasic activation, and larger engagement of central amygdala projecting cells with 25-Hz tonic light. These outcomes argue that the LC activation patterns differentially influence both target networks and behavior.

A novel G protein-biased agonist at the μ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase.

G protein-biased μ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased μ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced μ receptor desensitisation was probed using kinase inhibitors. Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased μ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular μ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid μ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. Compound 1 is a novel G protein-biased μ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Voltage modulates the effect of μ-receptor activation in a ligand-dependent manner.

Background and PurposeVarious GPCRs have been described as being modulated in a voltage‐dependent manner. Opioid analgesics act via activation of μ receptors in various neurons. As neurons are exposed to large changes in membrane potential, we were interested in studying the effects of depolarization on μ receptor signalling.Experimental ApproachWe investigated potential voltage sensitivity of μ receptors in heterologous expression systems (HEK293T cells) using electrophysiology in combination with Förster resonance energy transfer‐based assays. Depolarization‐induced changes in signalling were also tested in physiological rat tissue containing locus coeruleus neurons. We applied depolarization steps across the physiological range of membrane potentials.Key ResultsStudying μ receptor function and signalling in cells, we discovered that morphine‐induced signalling was strongly dependent on the membrane potential (VM). This became apparent at the level of G‐protein activation, G‐protein coupled inwardly rectifying potassium channel (Kir3.X) currents and binding of GPCR kinases and arrestin3 to μ receptors by a robust increase in signalling upon membrane depolarization. The pronounced voltage sensitivity of morphine‐induced μ receptor activation was also observed at the level of Kir3.X currents in rat locus coeruleus neurons. The efficacy of peptide ligands to activate μ receptors was not (Met‐enkephalin) or only moderately ([D‐Ala2, N‐Me‐Phe4, Gly5‐ol]‐enkephalin) enhanced upon depolarization. In contrast, depolarization reduced the ability of the analgesic fentanyl to activate μ receptors.Conclusion and ImplicationsOur results indicate a strong ligand‐dependent modulation of μ receptor activity by the membrane potential, suggesting preferential activity of morphine in neurons with high neuronal activity.

Inhibition of orexin receptor 1 contributes to the development of morphine dependence via attenuation of cAMP response element-binding protein and phospholipase Cβ3

Noradrenergic neurons of the locus coeruleus (LC) receive projection from hypothalamus orexinergic neurons and express orexin 1 receptor (Orx1). Orx in the locus coeruleus nucleus is involved in the development of morphine dependence. The downstream signaling of Orx contribution to the development of morphine dependence in LC neurons of morphine-dependent rats was studied. Therefore, we evaluated cyclic adenosine monophosphate (cAMP), cAMP response element-binding protein (CREB) and phospholipase Cβ3 (PLCβ3) levels by the application of immunohistochemistry. Results showed that cAMP, CREB and PLCβ3 levels were suppressed by the application of SB-334867 (as a selective Orx1 antagonist) in morphine-dependent rats. Our results unraveled that Orx1 blockade is involved in the development of morphine dependency through diminution of a variety of intracellular events including the cAMP, CREB and PLCβ3 levels in morphine-dependent rats. Furthermore, the Orx1 blockade could decrease the percentage of tyrosine hydroxylase (TH)+/CREB+ and TH+/PLCβ3+ neurons in LC of morphine-treated rats. It is concluded that the activation of Orx1 in LC nucleus might be involved in some intracellular changes in morphine dependent rats.

Maturation of NMDA receptor-mediated spontaneous postsynaptic currents in the rat locus coeruleus neurons.

During mammalian brain development, neural activity leads to maturation of glutamatergic innervations to locus coeruleus. In this study, fast excitatory postsynaptic currents mediated by N-methyl-d-aspartate (NMDA) receptors were evaluated to investigate the maturation of excitatory postsynaptic currents in locus coeruleus (LC) neurons. NMDA receptor-mediated synaptic currents in LC neurons were evaluated using whole-cell voltage-clamp recording during the primary postnatal weeks. This technique was used to calculate the optimum holding potential for NMDA receptor-mediated currents and the best frequency for detecting spontaneous excitatory postsynaptic currents (sEPSC). The optimum holding potential for detecting NMDA receptor-mediated currents was + 40 to + 50 mV in LC neurons. The frequency, amplitude, rise time, and decay time constant of synaptic responses depended on the age of the animal and increased during postnatal maturation. These findings suggest that most nascent glutamatergic synapses express functional NMDA receptors in the postnatal coerulear neurons, and that the activities of the neurons in this region demonstrate an age-dependent variation.

Decreased excitability of locus coeruleus neurons during hypercapnia is exaggerated in the streptozotocin-model of Alzheimer's disease

The locus coeruleus (LC) is a pontine nucleus important for respiratory control and central chemoreception. It is affected in Alzheimer's disease (AD) and alteration of LC cell function may account for respiratory problems observed in AD patients. In the current study, we tested the electrophysiological properties and CO2/pH sensitivity of LC neurons in a model for AD. Sporadic AD was induced in rats by intracerebroventricular injection of 2 mg/kg streptozotocin (STZ), which induces behavioral and molecular impairments found in AD. LC neurons were recorded using the patch clamp technique and tested for responses to CO2 (10% CO2, pH = 7.0). The majority (~60%) of noradrenergic LC neurons in adult rats were inhibited by CO2 exposure as indicated by a significant decrease in action potential (AP) discharge to step depolarizations. The STZ-AD rat model had a greater sensitivity to CO2 than controls. The increased CO2-sensitivity was demonstrated by a significantly stronger inhibition of activity during hypercapnia that was in part due to hyperpolarization of the resting membrane potential. Reduction of AP discharge in both groups was generally accompanied by lower LC network activity, depolarized AP threshold, increased AP repolarization, and increased current through a subpopulation of voltage-gated K+ channels (KV). The latter was indicated by enhanced transient KV currents particularly in the STZ-AD group. Interestingly, steady-state KV currents were reduced under hypercapnia, a change that would favor enhanced AP discharge. However, the collective response of most LC neurons in adult rats, and particularly those in the STZ-AD group, was inhibited by CO2.

P.528 Serotonergic 5-HT2A receptors are expressed in noradrenergic locus coeruleus neurons in rats

P.528 Serotonergic 5-HT2A receptors are expressed in noradrenergic locus coeruleus neurons in rats

PKC inhibitor reversed the suppressive effect of orexin-A on IPSCs of locus coeruleus neurons in naloxone-induced morphine withdrawal

The locus coeruleus (LC) as a target of addictive drugs receives a dense projection of orexinergic fibres from the lateral hypothalamus (LH) and is accordingly a candidate site for the expression of the somatic aspects of morphine withdrawal. Recently it has been shown that the inhibitory synaptic currents of LC neurons decrease partly through orexin type 1 receptors in the context of naloxone-induced morphine withdrawal; however, its cellular mechanism remains unclear. In this study, whole-cell patch clamp recordings of LC neurons in brainstem slices were used to investigate the impact of protein kinase C (PKC) on GABAergic inhibitory post-synaptic currents (IPSCs) in the context of naloxone-induced morphine withdrawal. Male Wistar rats (P14-P21) received morphine (20mg/kg, i.p.) daily for 7 consecutive days to induce morphine dependency. Our results showed that the application of PKC inhibitor (Go 6983; 1µM) alone did not decrease the probability of GABA release in the LC neurons of the morphine-treated rats in the presence of naloxone. Although, Go 6983 reversed the reduction of the amplitude of evoked IPSCs (eIPSCs) and spontaneous IPSCs (sIPSCs) frequency induced by orexin-A but did not change the sIPSCs amplitude. These results indicate that the suppressive effect of orexin-A on IPSCs is probably reversed by PKC inhibitor in the LC neurons of morphine-treated rats in the context of naloxone withdrawal.

Synergistic effect of orexin-glutamate co-administration on spontaneous discharge rate of locus coeruleus neurons in morphine-dependent rats

Various intrinsic and extrinsic factors can increase the spontaneous discharge rate of locus coeruleus (LC) neurons. Among the extrinsic ones, orexinergic neurons of the lateral hypothalamus (LH) send widespread projections to LC region. Accumulating evidence support the involvement of glutamate in mediating the excitatory effect of orexin-A on LC neurons. In addition, both orexinergic and glutamatergic systems have been shown to play critical roles in molecular changes underlying the development of morphine dependence. The present study was designed to investigate the interaction between orexin-A and glutamate in modulating the firing rate of LC neurons. Regarding the role of both orexinergic and glutamatergic systems in morphine dependence, this effect was also investigated in morphine dependent rats. For this purpose, spontaneous discharge rate of LC neurons was recorded using the whole-cell patch clamp recording method in presence of orexin-A, glutamate or orexin-A plus glutamate in acutely prepared brain slices. Results indicated that superfusion of either orexin-A or glutamate enhances the firing rate of LC neurons in both dependent and non-dependent rats. However, co-application of orexin-A and glutamate elicited a significant synergism in enhancement of firing rate only in morphine dependent animals. In conclusion, it seems that development of morphine dependence promotes adaptations in locus coeruleus neurons that potentiate the orexin-glutamate interaction.

The CO2/pH Chemosensitivity of Locus Coeruleus Neurons Is Increased in the Streptozotocin‐Model of Alzheimer's Disease

Patients with Alzheimer's disease (AD) develop pathological changes in the Locus Coeruleus (LC), an important pontine nucleus involved in respiratory control and central chemoreception. In addition, AD patients have breathing disturbances that may be related to LC dysfunction. Here, we tested the electrophysiological properties of LC neurons in a model for AD. Sporadic AD was induced in rats (6–7 weeks) by intracerebroventricular injection of streptozotocin (STZ; 2 mg/kg). After 14 days following injection, LC neurons were recorded using the patch clamp technique and tested for CO2 chemosensitivity (10% CO2, pH = 7.0).Hypercapnic exposure lowered current‐evoked spike discharge in the majority of LC neurons (~60%) when compared to baseline responses. The remaining cells either increased spiking (~20%) or did not respond (~20%) to CO2. Within the cell group that was inhibited by hypercapnia, baseline spike discharge to current injection was similar between control and STZ rats. These responses decreased significantly in both groups when exposed to 10% CO2 (bsl vs. 10% CO2: CTL, p=0.003, n=8 and STZ, p=0.001, n=9). The CO2‐induced decrease in spike discharge of the STZ group was significantly lower when compared to control (CTL vs. STZ, p=0.038), indicating greater sensitivity to hypercapnia. However, we observed no difference in resting membrane potential and input resistance (Ri, resistance across the cell membrane) between groups at baseline and CO2. Despite lack of difference between groups, within the STZ group Ri decreased significantly with hypercapnia (bsl, 126.5 ± 14.9 MΩ vs. 10% CO2, 98.4 ± 8.2 MΩ; p=0.01), indicating ion channel opening when exposed to CO2. Analysis of the current‐voltage relationship only showed a significant decrease in the steady state current under CO2 (bsl vs. 10% CO2: CTL, p=0.002, and STZ, p=0.001,), which had a similar magnitude in both groups. Close analysis of action potential (AP) shape (spike threshold, peak, slope, peak to anti‐peak) showed no difference between groups. Within the STZ rats, however, spike threshold was significantly shifted to more positive potentials when exposed to CO2 (−39.6 ± 1.9 mV vs −34.8 ± 2.2 mV, p=0.01). This shift in spike threshold likely contributes to the pronounced decrease in spike discharge in the STZ group during hypercapnic conditions.In summary, our data suggest that the majority of LC neurons in adult rats are inhibited by CO2. Further, the STZ‐treated group exhibits a greater sensitivity to CO2, possibly due to an increased spike threshold and opening of additional, yet unidentified membrane channels. A decreased excitability of LC neurons may play a role in the respiratory dysfunction observed in patients with AD.Support or Funding InformationFAPESP 2017/21750‐9 (MCV); ATSU seed money (TDO)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decrease of inhibitory synaptic currents of locus coeruleus neurons via orexin type 1 receptors in the context of naloxone-induced morphine withdrawal.

Acute opioid withdrawal syndrome is a series of neurological symptoms caused by the abrupt cessation of the chronic administration of opioids such as morphine. The locus coeruleus (LC) in the brain stem receives a dense projection of orexinergic fibers from the hypothalamus and is a candidate site for the expression of the somatic aspects of morphine withdrawal. Previous studies have shown that orexin-A contributes to the behavioral symptoms of naloxone-induced morphine withdrawal, partly by reducing the activity of GABAergic neurons, suggesting that orexin-A may negatively modulate fast GABAergic neurotransmission during morphine withdrawal. We used whole-cell patch-clamp recordings of LC neurons in brainstem slices to investigate the effect of orexin-A on bicuculline-sensitive GABAergic inhibitory postsynaptic currents (IPSCs) during naloxone-induced morphine withdrawal. Male Wistar rats (P14-P21) were given morphine (20mg/kg, i.p.) daily for seven consecutive days to create dependency on the drug. The application of naloxone (1µM) to brain slices of morphine-treated rats reduced the amplitude of evoked IPSCs (eIPSCs) as well as spontaneous IPSCs (sIPSCs) frequency but did not change sIPSCs amplitude. Orexin-A (100nM) significantly enhanced the suppressive effect of naloxone on eIPSCs amplitude and sIPSCs frequency but had no effect on the presence of the orexin type 1 receptor (OX1R) antagonist, SB-334867. Orexin-A alone had no significant effect on eIPSCs and sIPSCs in the absence of naloxone. In summary, our results show that orexin-A, via OX1R, potentiates the suppressive effect of naloxone on GABAergic IPSCs of LC neurons in morphine-treated rats. We conclude that orexins may have a critical role in regulating GABAergic neurotransmission to LC neurons during naloxone-induced morphine withdrawal.

Sex differences in morphine-induced trafficking of mu-opioid and corticotropin-releasing factor receptors in locus coeruleus neurons

The locus coeruleus (LC)-norepinephrine (NE) system is a key nucleus in which endogenous opioid and stress systems intersect to regulate the stress response. LC neurons of male rats become sensitized to stress following chronic morphine administration. Whether sex dictates this pattern of opioid-induced plasticity has not been demonstrated. Delineating the neurobiological adaptations produced by chronic opioids will enhance our understanding of stress vulnerability in opioid-dependent individuals, and may reveal how stress negatively impacts addiction recovery. In the present study, the effect of chronic morphine on the subcellular distribution of mu-opioid (MOR) and CRF receptors (CRFR) was investigated in the LC of male and female rats using immunoelectron microscopy. Results showed that placebo-treated females exhibited higher MOR and CRFR cytoplasmic distribution ratio when compared to placebo-treated males. Chronic morphine exposure induced a shift in the distribution of MOR immunogold-silver particles from the plasma membrane to the cytoplasm selectively in male LC neurons. Interestingly, chronic morphine exposure induced CRFR recruitment to the plasma membrane of both male and female LC neurons. These findings provide a potential mechanism by which chronic opioid administration increases stress vulnerability in males and females via an increase in surface availability of CRFR in LC neurons. However, our results also support the notion that cellular adaptations to chronic opioids differ across the sexes as redistribution of MOR following morphine exposure was only observed in male LC neurons.

Orexin A presynaptically decreases inhibitory synaptic transmission in rat locus coeruleus neurons

Locus coeruleus nucleus (LC) is a major noradrenergic nucleus in the brain. It receives dense orexinergic projections from lateral hypothalamus. Whilst it is known that orexin A increases firing rate of LC neurons, its effect on spontaneous and evoked inhibitory postsynaptic currents (sIPSCs and eIPSCs, respectively) has not been yet identified. In this research, we investigated the effect of orexin A on eIPSCs and sIPSCs in LC neurons. Whole-cell recordings revealed that orexin A suppresses eIPSCs amplitude in which this effect was blocked by an orexin type-1 receptors antagonist (SB-334867) and cannabinoid type-1 (CB1) receptors antagonist (AM251). Moreover, exposure of neurons to BAPTA (Ca2+ chelator) and U73122 (phospholipase C inhibitor) prevented orexin A-induced eIPSCs depression. On the other hand, orexin A increased pair pulse ratio and sIPSCs frequency but had no effect on sIPSCs amplitude. Our results revealed that eIPSCs suppression in the LC is mediated by CB1 receptor through a presynaptic mechanism.

Role of catalpol in ameliorating the pathogenesis of experimental autoimmune encephalomyelitis by increasing the level of noradrenaline in the locus coeruleus.

The endogenous neurotransmitter, noradrenaline, exerts anti-inflammatory and neuroprotective effects in vivo and in vitro. Reduced noradrenaline levels results in increased inflammation and neuronal damage. The primary source of noradrenaline in the central nervous system is tyrosine hydroxylase (TH)-positive neurons, located in the locus coeruleus (LC). TH is the rate-limiting enzyme for noradrenaline synthesis; therefore, regulation of TH protein expression and intrinsic enzyme activity represents the central means for controlling the synthesis of noradrenaline. Catalpol is an iridoid glycoside purified from Rehmannia glutinosa Libosch, which exerts a neuroprotective effect in multiple sclerosis (MS). The present study used an experimental mouse model of autoimmune encephalomyelitis to verify the neuroprotective effects of catalpol. Significant improvements in the clinical scores were observed in catalpol-treated mice. Furthermore, catalpol increased TH expression and increased noradrenaline levels in the spinal cord. In primary cultures, catalpol exerted a neuroprotective effect in rat LC neurons by increasing the noradrenaline output. These results suggested that drugs targeting LC survival and function, including catalpol, may be able to benefit patients with MS.

Repeated Administration of Amitriptyline in Neuropathic Pain: Modulation of the Noradrenergic Descending Inhibitory System.

The tricyclic antidepressant amitriptyline, the serotonin and noradrenaline reuptake inhibitor duloxetine, and gabapentinoids are first-line drugs for treatment of neuropathic pain. The analgesic effect of these drugs relates to brainstem-spinal descending noradrenergic systems. However, amitriptyline utilizes a variety of mechanisms for analgesia in neuropathic pain, and it is unclear which mechanism is most important. In the present study, we investigated the role of descending noradrenergic systems in the analgesic effect of these drugs for treatment of neuropathic pain. We also examined whether amitriptyline modifies the descending noradrenergic systems. Seven days after L5 spinal nerve ligation (SNL), rats received N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, 50 mg/kg) to degenerate noradrenergic fibers. The rats then received 5 daily intraperitoneal injections of amitriptyline (10 mg/kg), duloxetine (10 mg/kg), pregabalin (10 mg/kg), or gabapentin (50 mg/kg) from 21 days after SNL surgery. Paw withdrawal thresholds were determined to assess the effect of the drugs on hyperalgesia after SNL. To determine whether 5 daily injections of amitriptyline activated noradrenergic neurons in the locus coeruleus (LC) and spinal cord with or without DSP-4 treatment, we performed immunohistochemistry using antibodies for c-Fos and dopamine beta-hydroxylase (DβH). Five daily injections of amitriptyline, duloxetine, pregabalin, and gabapentin exerted antihyperalgesic effects in SNL rats (P < .001; estimated treatment effect of amitriptyline [99% confidence interval]: 59.9 [35.1-84.7] g). The antihyperalgesic effects of duloxetine, pregabalin, and gabapentin were reversed by pretreatment with DSP-4 (P < .001, respectively). However, antihyperalgesia was still observed after treatment of amitriptyline in SNL rats with DSP-4 pretreatment (P < .001, 59.7 [30.0-89.3] g), and this analgesic effect was not reversed by the α2-adrenoceptor antagonist idazoxan (30 μg). Additionally, 5 daily injections of amitriptyline increased the ratio of c-Fos-immunoreactive (IR) cells in noradrenergic LC neurons in SNL rats with or without DSP-4 pretreatment (P < .001, respectively). Five daily injections of amitriptyline increased DβH-IR in the LC and the spinal dorsal horn of SNL rats (P < .001, respectively). With DSP-4 pretreatment, DβH-IR was dramatically decreased with or without 5 daily injections of amitriptyline (P < .001). Five daily injections of amitriptyline produced antihyperalgesic effects against neuropathic pain despite suppression of noradrenergic descending inhibitory systems. Amitriptyline activated LC neurons and increased noradrenergic fibers density in SNL rats. These results suggest that amitriptyline could still produce analgesia under pathological dysfunction of the descending noradrenergic system. Amitriptyline may enhance the analgesic effect of drugs for neuropathic pain that require normal descending noradrenergic inhibition to produce analgesia, such as serotonin and noradrenaline reuptake inhibitors and gabapentinoids.

Enhancement of μ-opioid receptor desensitization by orexin-A in rat locus coeruleus neurons

Opioids have always been used in clinical practice for pain management. However, development of tolerance to their effects following long term administration, seriously restricts further clinical use of these drugs. In this regard, μ-opioid receptor (MOR) desensitization, as an initial step in development of opioid tolerance, is of particular significance. Previous studies support the involvement of orexinergic system in development of opioid tolerance. Locus coeruleus (LC) nucleus has been shown to modulate pain and development of tolerance. Opioid receptors (particularly μ) are densely expressed within the LC. Moreover, it receives widespread orexinergic inputs and orexin type 1 receptors (OX1Rs) are also highly expressed in this brain region. In the present study, the effect of orexin-A (OXA) on met-enkephalin (ME)-induced MOR desensitization was investigated in locus coeruleus neurons of male Wistar rats (2–3weeks of age). ME (30μM), as a potent MOR agonist, was applied for 10min and the outward K+ current was recorded using whole cell patch clamp recording. The percentage of decrease in ME-induced K+ current was considered as the degree of MOR desensitization. Results indicated that OXA (100nM) enhances ME-induced MOR desensitization via affecting OX1Rs in rat locus coeruleus neurons and this effect is mediated by a protein kinase C dependent mechanism within the LC. The activity of orexinergic system might potentiate the signaling pathways underlying opioid-induced receptor desensitization.