Activity Of Locus Coeruleus Neurons Research Articles

Hippocampal seizures differentially modulate locus coeruleus activity and result in consistent time-locked release of noradrenaline in rat hippocampus

The locus coeruleus (LC) is a small brainstem nucleus and is the sole source of noradrenaline in the neocortex, hippocampus and cerebellum. Noradrenaline is a powerful neuromodulator involved in the regulation of excitability and plasticity of large-scale brain networks. In this study, we performed a detailed assessment of the activity of locus coeruleus neurons and changes in noradrenergic transmission during acute hippocampal seizures evoked with perforant path stimulation, using state-of-the-art methodology. Action potentials of LC neurons, of which some were identified by means of optogenetics, were recorded in anesthetized rats using a multichannel high-density electrophysiology probe. The seizure-induced change in firing rate differed between LC neurons: 55% of neurons decreased in firing rate during seizures, while 28% increased their firing rate. Topographic analysis of multi-unit activity over the electrophysiology probe showed a topographic clustering of neurons that were inhibited or excited during seizures. Changes in hippocampal noradrenaline transmission during seizures were assessed using a fluorescent biosensor for noradrenaline, GRABNE2m, in combination with fiber photometry, in both anesthetized and awake rats. Although our neuronal recordings indicated both inhibition and excitation of LC neurons during seizures, a consistent release of noradrenaline was observed. Concentrations of noradrenaline increased at seizure onset and decreased during or shortly after the seizure. In conclusion, this study showed consistent but heterogeneous modulation of LC neurons and a consistent time-locked release of hippocampal noradrenaline during acute hippocampal seizures.

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.

Effects of sodium oxybate on hypocretin/orexin and locus coeruleus neurons.

Long-term use of sodium oxybate (SXB), (also called gamma-hydroxybutyrate [GHB]) attenuates the cataplexy and sleepiness of human narcolepsy. We had previously found that chronic opiate usage in humans and long-term opiate administration to mice significantly increased the number of detected hypocretin/orexin (Hcrt) neurons, decreased their size, and increased Hcrt level in the hypothalamus. We also found that opiates significantly decreased cataplexy in human narcoleptics as well as in narcoleptic mice and that cessation of locus coeruleus neuronal activity preceded and was tightly linked to cataplectic attacks in narcoleptic dogs. We tested the hypothesis that SXB produces changes similar to opiates and now report that chronic SXB administration significantly increased the size of Hcrt neurons, the reverse of what we had seen with opiates in humans and mice. Levels of Hcrt in the hypothalamus were nonsignificantly lower, in contrast to the significant increase in hypothalamic Hcrt level after opiates. SXB decreased tyrosine hydroxylase levels in the locus coeruleus, the major descending projection of the hypocretin system, also the reverse of what we saw with opioids. Therefore despite some similar effects on narcoleptic symptomatology, SXB does not produce anatomical changes similar to those elicited by opiates. Analysis of changes in other links in the cataplexy pathway might further illuminate SXB's mechanism of action on narcolepsy.

Vagus nerve stimulation parameters evoke differential neuronal responses in the locus coeruleus.

Vagus nerve stimulation (VNS) is used to treat drug-resistant epilepsy and depression, with additional applications under investigation. The noradrenergic center locus coeruleus (LC) is vital for VNS effects; however, the impact of varying stimulation parameters on LC activation is poorly understood. This study characterized LC activation across VNS parameters. Extracellular activity was recorded in rats' left LC while 11 VNS paradigms, utilizing variable frequencies and bursting characteristics, were pseudorandomly delivered to the left cervical vagus for five cycles. Neurons' change from baseline firing rate and timing response profiles were assessed. The proportion of neurons categorized as responders over 5 VNS cycles doubled in comparison to the first VNS cycle (p< 0.001) for all VNS paradigms, demonstrating an amplification effect. The percentage of positively consistent/positive responders increased for standard VNS paradigms with frequencies ≥10Hz and for bursting paradigms with shorter interburst intervals and more pulses per burst. The synchrony between pairs of LC neurons increased during bursting VNS but not standard paradigms. Also, the probability of evoking a direct response during bursting VNS was higher with longer interburst intervals and a higher number of pulses per burst. Standard paradigms between 10-30 Hz best positively activates LC with consistency to VNS while the best bursting paradigm to increase activity was 300 Hz, seven pulses per burst separated by 1s. Bursting VNS was effective in increasing synchrony between pairs of neurons, suggesting a common network recruitment originating from vagal afferents. These results indicate differential activation of LC neurons depending on the VNS parameters delivered.

Locus Coeruleus Neurons’ Firing Pattern Is Regulated by ERG Voltage-Gated K+ Channels

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG (ether-à-go-go-related gene) K+ channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

The effects of locus coeruleus stimulation on the BOLD global signal in TgF344‐AD rats

AbstractBackgroundFunctional connectivity of the brain as measured by the blood oxygen level dependent (BOLD) signal in fMRI is altered during Alzheimer’s disease (AD) progression. Common practice during preprocessing of fMRI data is removal of the global signal, defined as the averaged brain signal, by regression, removing major sources of noise while risking loss of neural activity. The locus coeruleus (LC), one of the first brain regions to accumulate hyperphosphorylated tau, contributes to brainwide neuromodulatory regulation. Therefore, we combined optogenetics and fMRI to study how LC activity affects the global signal in the TgF344‐AD rat model of AD, which overexpresses mutant human amyloid precursor protein and presenilin‐1.MethodsAt 2 months of age, TgF344‐AD rats and WT littermates received intra‐LC infusions of a ChR2‐mCherry virus under the noradrenergic specific PRSx8 promoter. 2‐4 weeks prior to fMRI, rats were implanted with an optic ferrule targeting the LC. Functional ChR2 expression and LC activation were confirmed by pupil dilation following optogenetic LC stimulation. At ∼6 months of age, rats underwent 10‐minute fMRI scans at baseline and during 2 Hz optogenetic stimulation. During scanning, rats were anesthetized with 1.3% isoflurane, intubated, and administered pancuronium (1.5 mg/kg*hr, s.c.). fMRI scans were gradient echo EPI (TR = 1.25 s) and obtained using a 9.4T Bruker Animal MRI scanner. Data were preprocessed using a customized pipeline. The voxel‐wise analysis was performed by correlating each voxel timecourse with the global signal via Pearson’s linear correlation.ResultsIn general, there were levels of slightly higher correlation to the global signal in the anterior medial region of the brain and bilaterally in the posterior region of the brain (Figure 1). The highest levels of correlation to the global signal were observed in the WT rats at baseline, but differences between groups were negligible.ConclusionNeither genotype nor optogenetic simulation profoundly affected global signal. These negative results may be explained by (1) the lack of profound forebrain AD‐like neuropathology in 6‐month TgF344‐AD rats, and (2) the 2 Hz optogenetic stimulation being insufficient to drive changes in global signal above the 1‐2 Hz baseline activity of LC neurons.

Brain‐wide Network Restructuring after Chemogenetic Locus Coeruleus Activation: Implications of Tonic Noradrenergic Activity for Neurodegeneration

Loss of noradrenergic neurons in a small brainstem nucleus, the locus coeruleus (LC), is a common characteristic in the development of age‐related neurodegenerative diseases such as Alzheimer's disease (AD) and other dementias. The noradrenergic system (NS) projects from the locus coeruleus (LC) throughout the brain to modulate neurophysiology for memory, learning and cognition, for regulation of sleep and circadian rhythm, and in response to stress and reward. Thus dysregulation of NS activity, i.e., blunted or tonic firing of LC neurons and its resultant effect on neural activity, is considered a significant factor in development and progression of neurodegenerative diseases as well as of psychiatric disorders. Our hypothesis is that tonic activation of LC neurons acutely discoordinates neuronal firing patterns across the brain, and ultimately provokes a prolonged abnormal brain state. We test this hypothesis via brain‐wide manganese‐enhanced MRI (MEMRI) in parallel with recordings of naturalistic behavior before, during, and long after chemogenetic (hM3Dq) stimulation of LC neurons. After systemic delivery, paramagnetic Mn(II) ions (0.3 mmol/kg, i.p.) accumulate in active neurons of awake‐behaving mice, highlighting neural activity by T1‐weighted MRI. Adult mice (3‐6 months) were subjected either to longitudinal MEMRI (n = 18) or to open field behavior (n = 8), ± hM3Dq expression; and ± designer drug – clozapine‐N‐oxide (CNO). For behavior naïve C57BL/6 mice were video recorded and behavioral parameters tracked by Noldus Ethovision XT15, before and after administration of CNO (5 mg/kg, i.p.). Mice then underwent stereotactic injection of CAV2‐PRS‐hM3D(Gq)‐mCherry targeting the LC (AP = ‐5.4, ML = ±0.80, DV = ‐3.80). At 4‐8 weeks after transfection the effect of CNO ± hM3Dq on LC neurons was monitored in live animals by pupillary light response. Pre‐ and post‐CNO behavior was again recorded. For MEMRI two groups ± hM3Dq were imaged before and after CNO. MEMRI images were analyzed for Mn(II)‐dependent intensity increases by statistical parametric mapping (SPM), segment‐wise cross‐correlation and network analysis. Transgene expression was confirmed post‐mortem by microscopy of mCherry in LC brain sections, which showed transgene expression in bilateral LC. Comparison of MEMRI segment‐wise intensities revealed changes of strength and direction of correlations between time points that also diffed between groups. Hierarchical clustering of these correlations revealed 40% increase in modularity, i.e., the number of distinct clusters, after CNO only in the hM3Dq transfected group. Together our data suggest discoordination of brain regions after LC‐NS activation. In neurodegenerative disease the loss of LC input could result in the opposite effects, while none‐the‐less leading to discoordination of brain‐wide activity underlying sleep disorders and memory disturbance. This brain‐wide imaging strategy with chemogenetic LC manipulation provides critical insight into NS modulation of neural activity under both normal and pathological conditions.

Somatostatin Neurons in the Mouse Pontine Nucleus Activate GABAA Receptor Mediated Synaptic Currents in Locus Coeruleus Neurons.

The pontine nuclei comprising the locus coeruleus (LC) and Barrington’s nucleus (BRN) amongst others form the neural circuitry(s) that coordinates arousal and voiding behaviors. However, little is known about the synaptic connectivity of neurons within or across these nuclei. These include corticotropin-releasing factor (CRF+) expressing neurons in the BRN that control bladder contraction and somatostatin expressing (SST+) neurons whose role in this region has not been discerned. To determine the synaptic connectivity of these neurons, we employed optogenetic stimulation with recordings from BRN and LC neurons in brain stem slices of channelrhodopsin-2 expressing SST or CRF neurons. Optogenetic stimulation of CRF+ BRN neurons of CrfCre;chr2-yfp mice had little effect on either CRF+ BRN neurons, CRF– BRN neurons, or LC neurons. In contrast, in SstCre;chr2-yfp mice light-activated inhibitory postsynaptic currents (IPSCs) were reliably observed in a majority of LC but not BRN neurons. The GABAA receptor antagonist, bicuculline, completely abolished the light-induced IPSCs. To ascertain if these neurons were part of the neural circuitry that controls the bladder, the trans-synaptic tracer, pseudorabies virus (PRV) was injected into the bladder wall of CrfCre;tdTomato or SstCre;tdTomato mice. At 68–72 h post-viral infection, PRV labeled neurons were present only in the BRN, being preponderant in CRF+ neurons with few SST+ BRN neurons labeled from the bladder. At 76 and 96 h post-virus injection, increased labeling was observed in both BRN and LC neurons. Our results suggest SST+ neurons rather than CRF+ neurons in BRN can regulate the activity of 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.

The role of glutamate transporter-1 in firing activity of locus coeruleus neurons and nociception in rats.

Locus coeruleus (LC) is considered to be the main source of norepinephrine in the central nervous system (CNS) and plays important role in relieving pain in the body. Changes in the activity of synaptic excitatory amino acid transporters (EAATs) would be an applicable way to regulate synaptic transmission in the LC. In the present study, we examined the role of astrocytic glutamate transporter-1 (GLT1) in the firing activity of LC neurons and the sensation of pain in rats. Male Wistar rats were divided into three control (CNT), ceftriaxone (CFT) and dihydrokainic acid (DHK) groups. Animals were given intraperitoneal injections for nine consecutive days after which the electrophysiological and behavioral experiments were performed to determine the single-unit activity of LC neurons and pain sensation. Results of this study revealed that CFT as a well-known up-regulator of GLT1 expression decreases the latency of pain sensation in rats but inhibition of GLT1 activity by DHK showed no significant effects. Furthermore, the results obtained by single-unit recording from LC showed a significant decrease in evoked response in CFT group compared to the CNT group. Therefore, this study suggests that GLT1 might be considered as a potential therapeutic target for pain modulation in the future.

Synchronous spiking associated with prefrontal high γ oscillations evokes a 5-Hz rhythmic modulation of spiking in locus coeruleus.

The brainstem noradrenergic locus coeruleus (LC) is reciprocally connected with the prefrontal cortex (PFC). Coupling between LC spiking and the depolarizing phase of slow (1-2 Hz) waves in PFC field potentials during sleep and anesthesia suggests that LC drives cortical state transition. Reciprocal LC-PFC connectivity should also allow interactions in the opposing (top-down) direction, but prior work has only studied prefrontal control over LC activity using electrical or optogenetic stimulation. Here, we describe the physiological characteristics of spontaneously occurring top-down LC-PFC interactions. We recorded LC multiunit activity (MUA) simultaneously with PFC single-unit and local field potential (LFP) activity in urethane-anesthetized rats. We observed cross-regional coupling between the phase of 5-Hz oscillations in LC-MUA and the power of PFC LFP 60-200 Hz high γ (hγ). Transient increases in PFC hγ power preceded peaks in the 5-Hz LC-MUA oscillation. Analysis of cross-regional transfer entropy demonstrated that the PFC hγ transients were predictive of a transient increase in LC-MUA. An ∼29 ms delay between these signals was consistent with the conduction velocity from the PFC to the LC. Finally, we showed that PFC hγ transients are associated with synchronized spiking of a subset (27%) of PFC single units. Our data suggest that PFC hγ transients may indicate the timing of the top-down excitatory input to LC, at least under conditions when LC neuronal population activity fluctuates rhythmically at 5 Hz. Synchronized PFC neuronal spiking that occurs during hγ transients may provide a previously unknown mode of top-down control over the LC.NEW & NOTEWORTHY The prefrontal cortex (PFC) is thought to control activity in the noradrenergic locus coeruleus (LC). Prior anatomical and prefrontal stimulation studies demonstrated the potential for PFC-LC interactions; however, it is unknown what types of PFC activity affect the LC. Here, we show that transient increases in PFC high γ power and associated changes in PFC unit-pair synchrony are a potential sign of top-down control over the LC.

Modulation of Noradrenergic and Serotonergic Systems by Cannabinoids: Electrophysiological, Neurochemical and Behavioral Evidence.

The main noradrenergic and serotonergic nuclei in the central nervous system (CNS) are the locus coeruleus (LC) and the dorsal raphe nucleus (DRN). These brain areas, located in the brainstem, play a pivotal role in the control of various functions and behaviors that are altered by cannabinoids (i.e., pain, arousal, mood, anxiety, or sleep-wake cycle). Anatomical, neurochemical, and functional data suggest that cannabinoids regulate both central noradrenergic and serotonergic neurotransmission. Thus, strong evidence has shown that the firing activity of LC and DRN monoamine neurons or the synthesis/release of noradrenaline (NA) and serotonin (5-HT) in the projection areas are all affected by cannabinoid administration. Herein, we propose that interaction between the endocannabinoid system and the noradrenergic-serotonergic systems could account for some of the anxiolytic, antidepressant, and antinociceptive effects of cannabinoids or the disruption of attention/sleep induced by these drugs.

Neuropathic pain increases spontaneous and noxious-evoked activity of locus coeruleus neurons

The noradrenergic locus coeruleus nucleus is an important station in both the ascending and descending pain regulatory pathways. These neurons discharge in tonic and phasic modes in response to sensory stimuli. However, few studies have set out to characterize the electrophysiological response of the locus coeruleus to noxious stimuli in conditions of neuropathic pain. Thus, the effects of mechanical nociceptive stimulation of the sciatic nerve area on spontaneous (tonic) and sensory-evoked (phasic) locus coeruleus discharge were studied by extracellular recording in anesthetized rats seven, fourteen and twenty-eight days after chronic constriction injury. Minor significant electrophysiological changes were found seven and fourteen days after nerve injury. However, alterations to the spontaneous activity in both the ipsilateral and contralateral locus coeruleus were found twenty-eight days after nerve constriction, as witnessed by an increase of burst firing incidence and irregular firing patterns. Furthermore, noxious-evoked responses were exacerbated in the contralateral and ipsilateral nucleus at twenty-eight days after injury, as were the responses evoked when stimulating the uninjured paw. In addition, mechanical stimulation of the hindpaw produced a significant sensitization of neuronal tonic activity after 28 days of neuropathy. In summary, long-term nerve injury led to higher spontaneous activity and exacerbated noxious-evoked responses in the locus coeruleus to stimulation of nerve-injured and even uninjured hindpaws, coinciding temporally with the development of depressive and anxiogenic-like behavior.

Orexin A induced increases in rat locus coeruleus neuronal activity are attenuated by systemic administration of OX1R and OX2R antagonists

Since the initial characterisation of the orexin/hypocretin system, the noradrenergic locus coeruleus (LC) has been a key focus due to its high expression of orexin receptors. Direct application of orexin A (OX-A) into the LC increases neural activity, and consequently arousal, attention and analgesia. We administered OX-A intracerebroventricularly to anaesthetised male CD® IGS rats and recorded spontaneous neural activity in the LC, blood pressure and heart rate. We then pre-treated with systemically administered orexin receptor 1 (OX1R) or orexin receptor 2 (OX2R) antagonists, SB-334867 and LSN2424100 respectively, and measured any changes in OX-A-induced effects. Central OX-A exposure dose dependently activated LC neurons over the period of 1 h: OX-A at 600 and 63 pmol, but not 20 pmol, increased neural activity. OX-A at 600 pmol, but not 63 or 20 pmol, altered blood pressure. OX-A did not alter heart rate at any of the doses tested. Pre-treatment with either OX1R or OX2R antagonist attenuated the OX-A-induced increase in LC firing with OX2R antagonism having a delayed onset. Neither antagonists altered LC firing on their own. We show that LC activation can be modulated by both OX1R and OX2R mechanisms, despite low OX2R expression in the LC. The LC is a key target brain nucleus in disease mechanisms that could be modulated by orexinergic pharmacology.

Electroacupuncture Ameliorates Acute Myocardial Ischemia: A Potential Role of the Locus Coeruleus.

The locus coeruleus (LC) is closely linked with cardiovascular disease. However, whether it mediates the alleviating effect of electroacupuncture (EA) on acute myocardial ischemia (AMI) remains unclear. A rat model of myocardial ischemia was established through occlusion of the left anterior descending coronary artery. Multichannel in vivo recording and other techniques were used to assess neurons in the LC, norepinephrine (NE) and dopamine (DA) levels in central and myocardial tissue, serum levels of inflammatory factors, and cardiac function. After induction of AMI, LC neuron activity increased and the central NE concentrations increased, while those of DA decreased. Moreover, the serum levels of high-sensitivity C-reactive protein (hs-CRP) increased, whereas those of interleukin-10 (IL-10) decreased. However, these effects were reversed by EA. Additionally, LC lesioning affected NE and DA levels in myocardial tissue and weakened the antimyocardial ischemic effect of EA. Collectively, our results indicated that LC is closely related to AMI and plays an important role in the antimyocardial ischemic effect of EA. This mechanism may be related to inhibition of LC neuron activity by EA, which inhibits the release of large amounts of hs-CRP and promotes that of IL-10 in the serum. Besides, after LC lesioning, EA may improve cardiac function by inhibiting the release of large amounts of NE and promoting the release of DA in myocardial tissue.

Calcium-induced calcium release in noradrenergic neurons of the locus coeruleus

The locus coeruleus (LC) is a nucleus within the brainstem that consists of norepinephrine-releasing neurons. It is involved in broad processes including cognitive and emotional functions. Understanding the mechanisms that control the excitability of LC neurons is important because they innervate widespread brain regions. One of the key regulators is cytosolic calcium concentration ([Ca2+]c), the increases in which can be amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. Although the electrical activities of LC neurons are regulated by changes in [Ca2+]c, the extent of CICR involvement in this regulation has remained unclear. Here we show that CICR hyperpolarizes acutely dissociated LC neurons of the rat and demonstrate the underlying pathway. When CICR was activated by extracellular application of 10 mM caffeine, LC neurons were hyperpolarized in the current-clamp mode of patch-clamp recording, and the majority of neurons showed an outward current in the voltage-clamp mode. This outward current was accompanied by increased membrane conductance, and its reversal potential was close to the K+ equilibrium potential, indicating that it is mediated by opening of K+ channels. The outward current was generated in the absence of extracellular calcium and was blocked when the calcium stores were inhibited by applying ryanodine. Pharmacological blockers indicated that it was mediated by Ca2+-activated K+ channels of the non-small conductance type. The application of caffeine increased [Ca2+]c, as visualized by fluorescence microscopy. These findings show CICR suppresses LC neuronal activity, and indicate its dynamic role in modulating the LC-mediated noradrenergic tone in the brain.

A brainstem-central amygdala circuit underlies defensive responses to learned threats

Norepinephrine (NE) plays a central role in the acquisition of aversive learning via actions in the lateral nucleus of the amygdala (LA) [1, 2]. However, the function of NE in expression of aversively-conditioned responses has not been established. Given the role of the central nucleus of the amygdala (CeA) in the expression of such behaviors [3–5], and the presence of NE axons projections in this brain nucleus [6], we assessed the effects of NE activity in the CeA on behavioral expression using receptor-specific pharmacology and cell- and projection-specific chemogenetic manipulations. We found that inhibition and activation of locus coeruleus (LC) neurons decreases and increases freezing to aversively conditioned cues, respectively. We then show that locally inhibiting or activating LC terminals in CeA is sufficient to achieve this bidirectional modulation of defensive reactions. These findings support the hypothesis that LC projections to CeA are critical for the expression of defensive responses elicited by conditioned threats.

Crosstalk between mitochondria, calcium channels and actin cytoskeleton modulates noradrenergic activity of locus coeruleus neurons.

Locus coeruleus (LC) is the name of a group of large sized neurons located at the brain stem, which provides the main source of noradrenaline to the central nervous system, virtually, innervating the whole brain. All noradrenergic signalling provided by this nucleus is dependent on an intrinsic pacemaker process. Our study aims to understand how noradrenergic neurons finely tune their pacemaker processes and regulate their activities. Here we present that mitochondrial perturbation in the LC from mice, inhibits spontaneous firing by a hyperpolarizing response that involves Ca2+ entry via L-type Ca2+ channels and the actin cytoskeleton. We found that pharmacological perturbation of mitochondria from LC neurons using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a dominant hyperpolarizing response when electrophysiological approaches were performed. Surprisingly, the CCCP-induced hyperpolarizing response was dependent on L-type Ca2+ channel-mediated Ca2+ entry, as it was inhibited by: the removal of extracellular Ca2+ ; the addition of Cd2+ ; nifedipine or nicardipine; but not by the intracellular dialysis with the Ca2+ chelator 1,2-Bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the latter indicating that the response was not because of a global change in [Ca2+ ]c but does not exclude action at intracellular microdomains. Further to this, the incubation of slices with cytochalasin D, an agent that depolymerises the actin cytoskeleton, inhibited the hyperpolarizing response indicating an involvement of the actin cytoskeleton. The data are consistent with the hypothesis that there is a crosstalk between mitochondria and L-type Ca2+ channels leading to modulation of noradrenergic neuronal activity mediated by the actin cytoskeleton. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

DREADD-mediated modulation of locus coeruleus inputs to mPFC improves strategy set-shifting.

Appropriate modification of behavior in response to our dynamic environment is essential for adaptation and survival. This adaptability allows organisms to maximize the utility of behavior-related energy expenditure. Modern theories of locus coeruleus (LC) function implicate a pivotal role for the noradrenergic nucleus in mediating switches between focused behavior during periods of high utility (exploit) versus disengagement of behavior and exploration of other, more rewarding opportunities. Two modes of activity in LC neurons have been characterized as elements in an Adaptive Gain Theory (AGT) of LC function. In this theory, during periods of accurate and focused behavior, LC neurons exhibit task-related phasic bursts. However, as behavioral utility wanes, phasic activity is suppressed and baseline (tonic) impulse activity increases to facilitate exploration. Our experiments sought to exogenously induce an elevated pattern of activity in LC neurons and their medial prefrontal cortical (mPFC) targets to test the tenets of the AGT. This theory posits that tonic activation immediately following a rule change should increase exploration and thereby improve performance on a set-shifting task. Indeed, DREADD mediated stimulation of LC terminals within mPFC decreased trials to reach criterion. However, this effect resulted from improved application of the new rule once the original rule is jettisoned rather than earlier disengagement from the old, ineffective strategy. Such improvements were not seen with global manipulation of LC, consistent with the view that LC-mediated exploration involves specific sub-circuits targeting mPFC. These findings extend our understanding of the role of LC in PFC and flexible behavior.