Neural circuit-selective, multiplexed pharmacological targeting of prefrontal cortex-projecting locus coeruleus neurons drives antinociception.

Selective manipulation of neural circuits using optogenetics and chemogenetics holds great translational potential but requires genetic access to neurons. Here, we demonstrate a general framework for identifying genetic tool-independent, pharmacological strategies for neural circuit-selective modulation. We developed an economically accessible calcium imaging-based approach for large-scale pharmacological scans of endogenous receptor-mediated neural activity. As a testbed for this approach, we used the mouse locus coeruleus due to the combination of its widespread, modular efferent neural circuitry and its wide variety of endogenously expressed GPCRs. Using machine learning-based action potential deconvolution and retrograde tracing, we identified an agonist cocktail that selectively inhibits medial prefrontal cortex-projecting locus coeruleus neurons. In vivo , this cocktail produces synergistic antinociception, consistent with selective pharmacological blunting of this neural circuit. This framework has broad utility for selective targeting of other neural circuits under different physiological and pathological states, facilitating non-genetic translational applications arising from cell type-selective discoveries.

A locus coeruleus to dorsal hippocampus pathway mediates cue-induced reinstatement of opioid self-administration in male and female rats.

Opioid use disorder is a chronic relapsing disorder encompassing misuse, dependence, and addiction to opioid drugs. Long term maintenance of associations between the reinforcing effects of the drug and the cues associated with its intake are a leading cause of relapse. Indeed, exposure to the salient drug-associated cues can lead to drug cravings and drug seeking behavior. The dorsal hippocampus (dHPC) and locus coeruleus (LC) have emerged as important structures for linking the subjective rewarding effects of opioids with environmental cues. However, their role in cue-induced reinstatement of opioid use remains to be further elucidated. In this study, we showed that chemogenetic inhibition of excitatory dHPC neurons during re-exposure to drug-associated cues significantly attenuates cue-induced reinstatement of morphine-seeking behavior. In addition, the same manipulation reduced reinstatement of sucrose-seeking behavior but failed to alter memory recall in the object location task. Finally, intact activity of tyrosine hydroxylase (TH) LC-dHPCTh afferents is necessary to drive cue induced reinstatement of morphine-seeking as inhibition of this pathway blunts cue-induced drug-seeking behavior. Altogether, these studies show an important role of the dHPC and LC-dHPCTh pathway in mediating cue-induced reinstatement of opioid seeking.

Endogenous opioids gate the locus coeruleus pain generator.

The locus coeruleus (LC) plays a paradoxical role in chronic pain. Although largely known as a potent source of endogenous analgesia, increasing evidence suggests injury can transform the LC into a chronic pain generator. We sought to clarify the role of this system in pain. Here, we show optogenetic inhibition of LC activity is acutely antinociceptive. Following long-term spared nerve injury, the same LC inhibition is analgesic - further supporting its pain generator function. To identify inhibitory substrates that may naturally serve this function, we turned to endogenous LC mu opioid receptors (LC-MOR). These receptors provide powerful LC inhibition and exogenous activation of LC-MOR is antinociceptive. We therefore hypothesized that endogenous LC-MOR-mediated inhibition is critical to how the LC modulates pain. Using cell type-selective conditional knockout and rescue of LC-MOR receptor signaling, we show these receptors bidirectionally regulate thermal and mechanical hyperalgesia - providing a functional gate on the LC pain generator.

An electrochemical approach for rapid, sensitive, and selective detection of dynorphin.

The endogenous opioid peptide systems are critical for analgesia, reward processing, and affect, but research on their release dynamics and function has been challenging. Here, we have developed microimmunoelectrodes (MIEs) for the electrochemical detection of opioid peptides using square-wave voltammetry. Briefly, a voltage is applied to the electrode to cause oxidation of the tyrosine residue on the opioid peptide of interest, which is detected as current. To provide selectivity to these voltammetric measurements, the carbon fiber surface of the MIE is coated with an antiserum selective to the opioid peptide of interest. To test the sensitivity of the MIEs, electrodes are immersed in solutions containing different concentrations of opioid peptides, and peak oxidative current is measured. We show that dynorphin antiserum-coated electrodes are sensitive to increasing concentrations of dynorphin in the attomolar range. To confirm selectivity, we also measured the oxidative current from exposure to tyrosine and other opioid peptides in solution. Our data show that dynorphin antiserum-coated MIEs are sensitive and selective for dynorphin with little to no oxidative current observed in met-enkephalin and tyrosine solutions. Additionally, we demonstrate the utility of these MIEs in an in vitro brain slice preparation using bath application of dynorphin as well as optogenetic activation of dynorphin release. Future work aims to use MIEs in vivo for real-time, rapid detection of endogenous opioid peptide release in awake, behaving animals.

Carbachol increases locus coeruleus activation by targeting noradrenergic neurons, inhibitory interneurons and inhibitory synaptic transmission.

The locus coeruleus (LC) consists of noradrenergic (NA) neurons and plays an important role in controlling behaviours. Although much of the knowledge regarding LC functions comes from studying behavioural outcomes upon administration of muscarinic acetylcholine receptor (mAChR) agonists into the nucleus, the exact mechanisms remain unclear. Here, we report that the application of carbachol (CCh), an mAChR agonist, increased the spontaneous action potentials (sAPs) of both LC-NA neurons and local inhibitory interneurons (LC I-INs) in acute brain slices by activating M1/M3 mAChRs (m1/3 AChRs). Optogenetic activation of LC I-INs evoked inhibitory postsynaptic currents (IPSCs) in LC-NA neurons that were mediated by γ-aminobutyric acid type A (GABAA ) and glycine receptors, and CCh application decreased the IPSC amplitude through a presynaptic mechanism by activating M4 mAChRs (m4 AChRs). LC-NA neurons also exhibited spontaneous phasic-like activity (sPLA); CCh application increased the incidence of this activity. This effect of CCh application was not observed with blockade of GABAA and glycine receptors, suggesting that the sPLA enhancement occurred likely because of the decreased synaptic transmission of LC I-INs onto LC-NA neurons by the m4 AChR activation and/or increased spiking rate of LC I-INs by the m1/3 AChR activation, which could lead to fatigue of the synaptic transmission. In conclusion, we report that CCh application, while inhibiting their synaptic transmission, increases sAP rates of LC-NA neurons and LC I-INs. Collectively, these effects provide insight into the cellular mechanisms underlying the behaviour modulations following the administration of muscarinic receptor agonists into the LC reported by the previous studies.

Comparing CLIF-C ACLF, CLIF-C ACLFlactate, and CLIF-C ACLF-D Prognostic Scores in Acute-on-Chronic Liver Failure Patients by a Single-Center ICU Experience.

Patients with liver cirrhosis have a higher risk of developing acute-on-chronic liver failure (ACLF). Poor prognosis with a high rate of short-term mortality leads to limited opportunities for further liver transplantation. Thus, precise prognostic evaluation of patients with ACLF is necessary before transplant surgery. In this study, a total of one hundred and thirty-five patients with ACLF admitted to the hepato-gastroenterologic intensive care unit (ICU) for intensive monitoring and treatment at Chang-Gung Memorial Hospital (CGMH, Linkou, Taiwan) were screened from November 2012 to April 2015 and tracked until April 2017. Three new prognostic scores of ACLF, including CLIF-C ACLF (Chronic Liver Failure Consortium Acute-on-chronic Liver Failure score), CLIF-C ACLF-D (CLIF-C ACLF Development score), and CLLF-C ACLFlactate (lactate-adjusted CLIF-C ACLF score) were compared. The primary outcome considered was overall mortality. Mortality predictions at 28, 90, 180, and 365 days were also calculated. By area under the receiver operating characteristic curve (AUROC) analysis, the CLIF-C ACLF and CLIF-C ACLF-D scores were superior to CLIF-C ACLFlactate scores in predicting 28-day mortality. The CLIF-C ACLF-D score had the highest AUROC in predicting overall mortality as well as at 90, 180, and 365 days. In conclusion, our study demonstrates that CLIF-C ACLF and CLIF-C ACLF-D scores are significant predictors of outcome in critical patients with liver cirrhosis and ACLF. The CLIF-C ACLF-D score may have a superior predictive power for the prediction of 3-month, 6-month, and one-year mortality.

Inhibitory interneurons regulate phasic activity of noradrenergic neurons in the mouse locus coeruleus and functional implications.

KEY POINTS: The locus coeruleus (LC) contains noradrenergic (NA) neurons that respond to novel stimuli in the environment with phasic activation to initiate an orienting response; phasic LC activation is also triggered by stimuli, representing the outcome of task-related decision processes, to facilitate ensuing behaviours and help optimize task performance. Here, we report that LC-NA neurons exhibit bursts of action potentials in vitro resembling phasic LC activation in vivo, and the activity is gated by inhibitory interneurons (I-INs) located in the peri-LC. We also observe that inhibition of peri-LC I-INs enhances prepulse inhibition and axons from cortical areas that play important roles in evaluating the cost/reward of a stimulus synapse on both peri-LC I-INs and LC-NA neurons. The results help us understand the cellular mechanisms underlying the generation and regulation of phasic LC activation with a focus on the role of peri-LC I-INs. ABSTRACT: Noradrenergic (NA) neurons in the locus coeruleus (LC) have global axonal projection to the brain. These neurons discharge action potentials phasically in response to either novel stimuli in the environment to initiate an orienting behaviour or stimuli representing the outcome of task-related decision processes to facilitate ensuing behaviours and help optimize task performance. Nevertheless, the cellular mechanisms underlying the generation and regulation of phasic LC activation remain unknown. We report here that LC-NA neurons recorded in brain slices exhibit bursts of action potentials that resembled the phasic activation-pause profile observed in animals. The activity was referred to as phasic-like activity (PLA) and was suppressed and enhanced by blocking excitatory and inhibitory synaptic transmissions, respectively. These results suggest the existence of a local circuit to drive PLA, and the activity could be regulated by the excitatory-inhibitory balance of the circuit. In support of this notion, we located a population of inhibitory interneurons (I-INs) in the medial part of the peri-LC that exerted feedforward inhibition of LC-NA neurons through GABAergic and glycinergic transmissions. Selective inhibition of peri-LC I-INs with chemogenetic methods could enhance PLA in brain slices and increase prepulse inhibition in animals. Moreover, axons from the orbitofrontal and prelimbic cortices, which play important roles in evaluating the cost/reward of a stimulus, synapse on both peri-LC I-INs and LC-NA neurons. These observations demonstrate functional roles of peri-LC I-INs in integrating inputs of the frontal cortex onto LC-NA neurons and gating the phasic LC output.

Extracellular Signal-Regulated Kinases Mediate an Autoregulation of GABAB-Receptor-Activated Whole-Cell Current in Locus Coeruleus Neurons.

The norepinephrine-releasing neurons in the locus coeruleus (LC) are well known to regulate wakefulness/arousal. They display active firing during wakefulness and a decreased discharge rate during sleep. We have previously reported that LC neurons express large numbers of GABAB receptors (GABABRs) located at peri-/extrasynaptic sites and are subject to tonic inhibition due to the continuous activation of GABABRs by ambient GABA, which is significantly higher during sleep than during wakefulness. In this study, we further showed using western blot analysis that the activation of GABABRs with baclofen could increase the level of phosphorylated extracellular signal-regulated kinase 1 (ERK1) in LC tissue. Recordings from LC neurons in brain slices showed that the inhibition of ERK1/2 with U0126 and FR180204 accelerated the decay of whole-cell membrane current induced by prolonged baclofen application. In addition, the inhibition of ERK1/2 also increased spontaneous firing and reduced tonic inhibition of LC neurons after prolonged exposure to baclofen. These results suggest a new role of GABABRs in mediating ERK1-dependent autoregulation of the stability of GABABR-activated whole-cell current, in addition to its well-known effect on gated potassium channels, to cause a tonic current in LC neurons.