Locus coeruleus: From global projection system to adaptive regulation of behavior.

The brainstem nucleus locus coeruleus (LC) is a major source of norepinephrine (NE) projections throughout the CNS. This important property was masked in very early studies by the inability to visualize endogenous monoamines. The development of monoamine histofluorescence methods by Swedish scientists led to a plethora of studies, including a paper published in Brain Research by Loizou in 1969. That paper was highly cited (making it a focal point for the 50th anniversary issue of this journal), and helped to spark a large and continuing set of investigations to further refine our understating of the LC-NE system and its contribution to brain function and behavior. This paper very briefly reviews the ensuing advances in anatomical, physiological and behavioral aspects of the LC-NE system. Although its projections are ubiquitously present throughout the CNS, recent studies find surprising specificity within the organizational and operational domains of LC neurons. These and other findings lead us to expect that future work will unmask additional features of the LC-NE system and its roles in normative and pathological brain and behavioral processes. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Atomoxetine modulates spontaneous and sensory-evoked discharge of locus coeruleus noradrenergic neurons.

Atomoxetine (ATM) is a potent norepinephrine (NE) uptake inhibitor and increases both NE and dopamine synaptic levels in prefrontal cortex, where it is thought to exert its beneficial effects on attention and impulsivity. At the behavioral level, ATM has been shown to cause improvements on the measures of executive functions, such as response inhibition, working memory and attentional set shifting across different species. However, the exact mechanism of action for ATM's effects on cognition is still not clear. One possible target for the cognitive enhancing effects of ATM is the noradrenergic locus coeruleus (LC), the only source of NE to key forebrain areas such as cerebral cortex and hippocampus. Although it is known that ATM increases NE availability overall by blocking reuptake of NE, the effects of this agent on impulse activity of LC neurons have not been reported. Here, the effect of ATM (0.1-1 mg/kg, ip) on NE-LC neurons was investigated by recording extracellular activity of LC neurons in isoflurane-anesthetized rats. ATM caused a significant decrease of the tonic activity of LC single-units, although leaving intact the sensory-evoked excitatory component of LC phasic response. Moreover, the magnitude of the inhibitory component of LC response to paw stimulation was increased after 1 mg/kg of ATM and its duration was prolonged at 0.3 mg/kg. Together, these effects of ATM produced an increase in the phasic-to-tonic ratio of LC phasic response to sensory stimulation. ATM also modulated the average sensory-evoked local field potential (LFP) and spike-field coherence in LC depending on the dose tested. The lower dose (0.1 mg/kg) significantly decreased early positive and negative components of the sensory-evoked LFP response. Higher doses (0.3-1 mg/kg) initially increased and then decreased the amplitude of components of the evoked fields, whereas the spike-field coherence was enhanced by 1 mg/kg ATM across frequency bands. Finally, coherence between LC fields and EEG signals was generally increased by 1 mg/kg ATM, whereas 0.1 and 0.3 mg/kg respectively decreased and increased coherence values in specific frequency bands. Taken together these results suggest that ATM effects on LC neuronal activity are dose-dependent, with different doses affecting different aspects of LC firing. This modulation of activity of LC-NE neurons may play a role in the cognitive effects of ATM. This article is part of a Special Issue entitled 'Cognitive Enhancers'.

Light deprivation damages monoamine neurons and produces a depressive behavioral phenotype in rats.

Light is an important environmental factor for regulation of mood. There is a high frequency of seasonal affective disorder in high latitudes where light exposure is limited, and bright light therapy is a successful antidepressant treatment. We recently showed that rats kept for 6 weeks in constant darkness (DD) have anatomical and behavioral features similar to depressed patients, including dysregulation of circadian sleep-waking rhythms and impairment of the noradrenergic (NA)-locus coeruleus (LC) system. Here, we analyzed the cell viability of neural systems related to the pathophysiology of depression after DD, including NA-LC, serotoninergic-raphe nuclei and dopaminergic-ventral tegmental area neurons, and evaluated the depressive behavioral profile of light-deprived rats. We found increased apoptosis in the three aminergic systems analyzed when compared with animals maintained for 6 weeks in 12:12 light-dark conditions. The most apoptosis was observed in NA-LC neurons, associated with a significant decrease in the number of cortical NA boutons. Behaviorally, DD induced a depression-like condition as measured by increased immobility in a forced swim test (FST). DD did not appear to be stressful (no effect on adrenal or body weights) but may have sensitized responses to subsequent stressors (increased fecal number during the FST). We also found that the antidepressant desipramine decreases these neural and behavioral effects of light deprivation. These findings indicate that DD induces neural damage in monoamine brain systems and this damage is associated with a depressive behavioral phenotype. Our results suggest a mechanism whereby prolonged limited light intensity could negatively impact mood.

Elevations of FosB in the nucleus accumbens during forced cocaine abstinence correlate with divergent changes in reward function.

Dysregulation of hedonic processing, in which seeking of drug reward becomes more desirable than seeking natural rewards, like food, sex, and novelty, is a consequence of chronic drug exposure and potentially leads to escalating drug usage and addiction. Here, we investigated the effects of chronic cocaine treatment (10 days of escalating doses of cocaine, 10-30 mg/kg) and multiple forced abstinence periods (2, 3 or 5 weeks) on the acute rewarding properties of either cocaine (10 mg/kg) or novel-objects using the conditioned place preference procedure. Following all cocaine withdrawal periods, cocaine preference was significantly elevated while novel object preference was abolished compared with saline-treated rats. At the earliest withdrawal period, these behavioral changes were accompanied by elevations in FosB-like immunoreactive staining in the basolateral amygdala (BLA) and nucleus accumbens shell (NAc-Sh) and core (NAc-C). FosB staining in all three brain areas correlated positively with cocaine preference, but negatively with novelty preference. After 5 weeks of withdrawal, FosB staining was only elevated in the NAc-Sh and again correlated positively with elevated cocaine preference but negatively with decreased novelty preference. These data indicate that alterations in the expression of FosB-like transcription factors in the NAc can predict the dysregulation of hedonic processing that occurs during protracted withdrawal from cocaine.

Glutamate-associated plasticity in the ventral tegmental area is necessary for conditioning environmental stimuli with morphine.

We sought to determine if plasticity in the ventral tegmental area (VTA) of the midbrain is involved in learning to associate morphine exposure with a specific environment. For this, we tested whether activation of glutamate receptors and protein kinase A is needed for the acquisition and expression of a morphine-conditioned place preference (CPP). Rats received bilateral microinjections of either the NMDA antagonist AP5 (0.48 nmol/0.3 microl), the AMPA antagonist CNQX (0.21 nmol/0.3 microl), or vehicle into the VTA prior to each of three morphine-conditioning sessions. Both the AMPA and NMDA receptor antagonists blocked the development of morphine CPP when given into the VTA but not when given outside the VTA. In similar studies the protein kinase A (PKA) inhibitor, Rp-cAMPS (13 nmol/0.3 microl), blocked the acquisition of morphine CPP when given into the VTA immediately after morphine conditioning. In separate experiments, glutamate antagonists, or Rp-cAMPS, immediately prior to the preference test blocked the expression of morphine CPP when microinjected into the VTA. These data indicate that the VTA is an important site for synaptic modifications involved in the learning and memory of environmental cues predicting reward, and that glutamate input and PKA activation are crucial to this process.

Potent regulation of midbrain dopamine neurons by the bed nucleus of the stria terminalis.

Recent studies have revealed an important role of the ventrolateral (subcommissural) aspect of the bed nucleus of the stria terminalis (vBNST) in motivational aspects of drug abuse (Delfs et al., 2000). Dopaminergic (DA) neurons in the ventral tegmental area (VTA) have also long been linked to motivation and drug abuse (Koob and Le Moal, 2001). The present study tested whether activity in the vBNST influences discharge of midbrain DA neurons. Responses of DA neurons in the VTA to activation of the vBNST were characterized in anesthetized rats using extracellular recording techniques. Electrical or chemical [10-50 mm glutamate (Glu)] stimulation of the vBNST consistently activated DA cells (122% increase in activity with 50 mm Glu). However, stronger chemical stimulation of the vBNST (100 mm Glu) completely inactivated DA neurons. In addition, apomorphine restored the activity of DA neurons that were inactivated by 100 mm Glu stimulation of the vBNST, indicating possible depolarization blockade of DA cells by vBNST activity. These findings reveal that the vBNST exerts a strong excitatory influence on DA neurons. Also striking was the finding that chemical stimulation (50 mm Glu) of the vBNST yielded long-lasting oscillatory activity (>15 min) in VTA DA neurons. These results indicate that the vBNST can generate long-lasting alterations in the activity of DA neurons in vivo.

Preference for a cocaine-associated environment is attenuated by augmented accumbal serotonin in cocaine withdrawn rats.

RATIONALE: Recent studies have found decreased serotonin (5-HT) transmission within the nucleus accumbens following withdrawal from chronic cocaine. OBJECTIVE: We sought to investigate whether increasing brain 5-HT levels would decrease behavioral responses that occur following cocaine withdrawal, namely increased preference for a cocaine environment and anxiety. METHODS: The conditioned place preference and the defensive burying paradigms were used to measure the behavioral responses that occur 1 week following cocaine withdrawal. RESULTS: We show that pharmacological agents that increase 5-HT transmission (sertraline or 5-hydoxytryptophan, 5-HTP) abolish the preference of subchronically cocaine-treated, abstinent rats for a cocaine-associated environment. Similar results were seen when sertraline was microinjected into the nucleus accumbens. Conversely, rats acutely conditioned with cocaine showed an increased preference for a cocaine-associated environment when pretreated with these drugs. Sertraline also decreased the heightened anxiety-like behaviors found in subchronically treated cocaine rats. CONCLUSIONS: These results indicate that drugs that augment 5-HT function may reduce the desire for cocaine following cocaine withdrawal, and thus facilitate cocaine abstinence in dependent subjects.

A neural circuit for circadian regulation of arousal.

An unknown aspect of behavioral state regulation is how the circadian oscillator of the suprachiasmatic nucleus (SCN) regulates sleep and waking. In this report, we describe the necessary elements for a circuit that provides circadian regulation of arousal. Trans-synaptic retrograde tracing revealed a prominent indirect projection from the SCN to the noradrenergic nucleus locus coeruleus (LC), a brain arousal system. Double-labeling experiments revealed several possible links between the SCN and the LC, including the dorsomedial (DMH) and paraventricular hypothalamic nuclei (PVN), as well as medial and ventrolateral pre-optic areas. Lesion studies confirmed that the DMH is a substantial relay in this circuit. Next, neurophysiology experiments revealed circadian variations in LC impulse activity. Lesions of the DMH eliminated these circadian changes in LC activity, confirming the functionality of the SCN-DMH-LC circuit. These results reveal mechanisms for regulation of circadian and sleep-waking functions.

Local opiate withdrawal in locus coeruleus neurons in vitro.

Noradrenergic neurons of the brain nucleus locus coeruleus (LC) become hyperactive during opiate withdrawal. It has been uncertain to what extent such hyperactivity reflects changes in intrinsic properties of these cells. The effects of withdrawal from chronic morphine on the activity of LC neurons were studied using intracellular recordings in rat brain slices. LC neurons in slices from chronically morphine-treated rats exhibited more than twice the frequency of spontaneous action potentials after naloxone compared with LC neurons from control rats. However, after naloxone treatment, the resting membrane potential (MP) of LC neurons from dependent rats was not significantly different from that in control rats. Neither resting MP nor spontaneous discharge rate (SDR) was altered by naloxone in LC neurons from control rats. Neither kynurenic acid nor a cocktail of glutamate and GABA antagonists (6-cyano-7-nitroquinoxalene-2,3-dione + 2-amino-5-phosphonopentanoic acid + bicuculline) blocked the hyperactivity of LC neurons precipitated by naloxone in slices from morphine-dependent rats. The effects of ouabain on MP and SDR were similar in LC neurons from control and morphine-dependent rats. These results indicate that an adaptive change in glutamatergic or GABAergic synaptic mechanisms or altered Na/K pump activity does not underlie the withdrawal-induced activation of LC neurons in vitro. Specific inhibitors of protein kinase A [Rp-cAMPS or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89)] partially suppressed the withdrawal hyperactivity of LC neurons, and activators of cAMP (forskolin) or protein kinase A (Sp-cAMPS) increased the discharge rate of LC neurons from control rats. These results suggest that upregulation of cAMP-dependent protein kinase A during chronic morphine treatment is involved in the withdrawal-induced hyperactivity of LC neurons.

Augmented accumbal serotonin levels decrease the preference for a morphine associated environment during withdrawal.

Recent studies have found that acute morphine administration increases serotonin (5-HT) transmission within the nucleus accumbens and other forebrain regions. In contrast, 5-HT transmission is depressed during withdrawal from chronic morphine. We show that pharmacological agents that increase brain 5-HT levels (fluoxetine or 5-hydoxytryptophan, 5-HTP) abolish the preference of chronically morphine-treated, withdrawn rats for a morphine-associated environment. Similar results were seen when fluoxetine was microinjected into the nucleus accumbens. Conversely, rats given morphine acutely showed an enhanced preference for a morphine-associated environment when pretreated with these agents. Fluoxetine also decreased the heightened anxiety found in morphine withdrawn rats. The results of our study indicate that drugs that augment 5-HT levels may reduce the desire for morphine during withdrawal.

Use of pseudorabies virus to delineate multisynaptic circuits in brain: opportunities and limitations.

Transsynaptic tracing with live virus is a powerful tool that has been used extensively to analyze central efferents that regulate peripheral targets. More recently, investigators have begun to use this new methodology with central injections to identify circuit anatomy within the brain. Although transsynaptic tracing with peripheral injection of pseudorabies virus has been extensively characterized, several methodological issues related to central application of this tracer have not been addressed. Here, we review the following issues relevant to the use of pseudorabies virus (PRV; Bartha strain) in experiments involving injection of virus into rat brain: (i) factors that determine the zone of viral uptake; (ii) uptake of pseudorabies virus by fibers of passage; (iii) viral invasion of the brain after leakage of virus into the brain ventricles; (iv) considerations for double labeling for PRV with peptides and neurotransmitters; (v) use of PRV with conventional retrograde tracers to anatomically identify relays in a multisynaptic pathway; and (vi) transport of PRV throughout the dendritic tree as a means of identifying inputs to distal dendrites. Collectively, the data demonstrate that PRV provides a powerful means of dissecting the synaptology of CNS circuitry when appropriate controls are incorporated into the experimental design. A set of recipes for various procedures are included at the end of this article.

Expression of fos-related antigens in the nucleus accumbens during opiate withdrawal and their attenuation by a D2 dopamine receptor agonist.

Previous studies from this laboratory indicated that D2 dopamine (DA) receptors within the nucleus accumbens (NAc) are important for regulating somatic signs of opiate withdrawal. The present study measured the expression of Fos-related antigens (FRAs) within the NAc during opiate withdrawal to determine whether decreases in somatic withdrawal signs produced by a D2 receptor agonist are accompanied by related changes in accumbens neuronal activity. In an initial experiment, quantitative analyses of FRA immunoreactivity revealed increases in the number of FRA-positive cells throughout the NAc of opiate dependent animals undergoing naltrexone-precipitated withdrawal relative to dependent or non-dependent animals that did not experience withdrawal. A second experiment showed that somatic signs and FRA expression within the NAc could each be attenuated when the D2 agonist propylnorapomorphine (NPA; 0.1 or 0.3 mg/kg, i.p.) was administered prior to naltrexone-precipitated withdrawal. These findings suggest that D2 regulation of neuronal activity within the NAc may be important for the expression of opiate withdrawal symptoms.

Behavioral activation induced by D(2)-like receptor stimulation during opiate withdrawal.

Withdrawal is a potent motivator of drug-seeking behavior in human opiate addicts. Paradoxically, opiate withdrawal reduces dopamine release and suppresses behavioral responding in several animal models of addiction. These findings pose critical questions about how a withdrawal state that depresses dopaminergic and behavioral functioning contributes to drug seeking. This study addressed this issue by investigating factors that increase behavioral activity during opiate withdrawal. Initial experiments revealed that the D(2)-like agonists propylnorapomorphine HCl (NPA; 0.05-0.4 mg/kg, i. p.) and quinpirole (0.2-0.4 mg/kg, s.c.) each produced strong locomotor activating effects during opiate withdrawal that were not apparent in the absence of withdrawal. Concurrent stereotypy ratings indicated that these effects of NPA and quinpirole during withdrawal were not an indirect consequence of changes in the stereotypy-inducing effects of these drugs. Subsequent experiments showed that locomotion was not increased when opiate withdrawal was induced in the presence of the D(1)-like agonist SKF 38393 (1.0-8.0 mg/kg, i.p.), that the locomotor activation produced by NPA during withdrawal could be attenuated by the D(2)-like antagonist eticlopride (0.1-0.2 mg/kg, i.p.), and that locomotor activating effects of NPA could be observed when withdrawal was induced by extracting the implanted morphine pellets, but not when the NPA was given after naltrexone antagonism of acute morphine treatment in nondependent rats. These findings indicate that opiate withdrawal regulates the behavioral impact of D(2)-like receptor stimulation so that locomotion is markedly increased when these receptors are stimulated during periods of withdrawal. This potentiation may be important for facilitating behavioral responses during periods of opiate detoxification.

Hypocretin/orexin depolarizes and decreases potassium conductance in locus coeruleus neurons.

Recent studies demonstrated that noradrenergic locus coeruleus (LC) neurons are a particularly strong target of the novel neuropeptide, hypocretin (orexin). The present study sought to elucidate the action of hypocretin-B (HCRT) on LC neurons recorded intracellularly in rat brain slices. Bath (1.0 microM) or local puff application (50-100 microM in pipette) of HCRT depolarized LC neurons in rat brain slices and increased their spontaneous discharge rate. Depolarization evoked by HCRT was persistent in the presence of tetrodotoxin (TTX, 1 microM) and Co2+ (1 mM), indicating that HCRT directly activated LC neurons, and that its effect on the postsynaptic cell was not due to activation of TTX-sensitive sodium channels or Co2+-sensitive calcium channels. The apparent input resistance was significantly increased in the majority of LC neurons during the HCRT-evoked depolarization. Moreover, the HCRT-evoked depolarization was decreased in amplitude with hyperpolarization of membrane. The present results indicate that decreased potassium conductance is involved in the effect of HCRT on LC neurons.

Noradrenaline in the ventral forebrain is critical for opiate withdrawal-induced aversion.

Cessation of drug use in chronic opiate abusers produces a severe withdrawal syndrome that is highly aversive, and avoidance of withdrawal or associated stimuli is a major factor contributing to opiate abuse. Increased noradrenaline in the brain has long been implicated in opiate withdrawal, but it has not been clear which noradrenergic systems are involved. Here we show that microinjection of beta-noradrenergic-receptor antagonists, or of an alpha2-receptor agonist, into the bed nucleus of the stria terminalis (BNST) in rats markedly attenuates opiate-withdrawal-induced conditioned place aversion. Immunohistochemical studies revealed that numerous BNST-projecting cells in the A1 and A2 noradrenergic cell groups of the caudal medulla were activated during withdrawal. Lesion of these ascending medullary projections also greatly reduced opiate-withdrawal-induced place aversion, whereas lesion of locus coeruleus noradrenergic projections had no effect on opiate-withdrawal behaviour. We conclude that noradrenergic inputs to the BNST from the caudal medulla are critically involved in the aversiveness of opiate withdrawal.

Hypocretin (orexin) activation and synaptic innervation of the locus coeruleus noradrenergic system.

Hypocretin has been identified as a regulator of metabolic and endocrine systems. Several brain regions involved in the central regulation of autonomic and endocrine processes or attention are targets of extensive hypocretin projections. The most dense arborization of hypocretin axons in the brainstem was detected in the locus coeruleus (LC). Multiple labeling immunocytochemistry revealed a massive synaptic innervation of catecholaminergic LC cells by hypocretin axon terminals in rats and monkeys. In both species, all tyrosine hydroxylase-immunopositive cells in the LC examined by electron microscopy were found to receive asymmetrical (excitatory) synaptic contacts from multiple axons containing hypocretin. In parallel electrophysiological studies with slices of rat brain, all LC cells showed excitatory responses to the hypocretin-2 peptide. Hypocretin-2 uniformly increased the frequency of action potentials in these cells, even in the presence of tetrodotoxin, indicating that receptors responding to hypocretin were expressed in LC neurons. Two mechanisms for the increased firing rate appeared to be a reduction in the slow component of the afterhyperpolarization (AHP) and a modest depolarization. Catecholamine systems in other parts of the brain, including those found in the medulla, zona incerta, substantia nigra or olfactory bulb, received significantly less hypocretin input. Comparative analysis of lateral hypothalamic input to the LC revealed that hypocretin-containing axon terminals were substantially more abundant than those containing melanin-concentrating hormone. The present results provide evidence for direct action of hypothalamic hypocretin cells on the LC noradrenergic system in rats and monkeys. Our observations suggest a signaling pathway via which signals acting on the lateral hypothalamus may influence the activity of the LC and thereby a variety of CNSfunctions related to noradrenergic innervation, including vigilance, attention, learning, and memory. Thus, the hypocretin innervation of the LC may serve to focus cognitive processes to compliment hypocretin-mediated activation of autonomic centers already described.

Role of locus coeruleus in attention and behavioral flexibility.

Previous findings have implicated the noradrenergic locus coeruleus (LC) system in functions along the dimension of arousal or attention. It has remained uncertain what role this system has in attention, or what mechanisms may be involved. We review our recent work examining activity of LC neurons in monkeys performing a visual discrimination task that requires focused attention. Results indicate that LC cells exhibit phasic or tonic modes of activity, that closely correspond to good or poor performance on this task, respectively. A computational model was used to simulate these results. This model predicts that alterations in electrotonic coupling among LC cells may produce the different modes of activity and corresponding differences in performance. This model also indicates that the phasic mode of LC activity may promote focused or selective attention, whereas the tonic mode may produce a state of high behavioral flexibility or scanning attentiveness. The implications of these results for clinical disorders such as attention-deficit hyperactivity disorder, stress disorders, and emotional and affective disorders are discussed.

Characterization of transsynaptic tracing with central application of pseudorabies virus.

Although transsynaptic tracing with peripheral injection of pseudorabies virus (PRV) has been extensively characterized, several methodological issues related to central application of this tracer have not been addressed. In the present study, we addressed the following three issues by using microinjection of a cocktail containing PRV (Bartha strain) and cholera toxin subunit B (CTb) into different sites in the rat brain. First, we estimated PRV diffusion by examining injection sites at different times after application. Second, we tested whether PRV is taken up by fibers of passage following injections into the olivocerebellar pathway. Third, we developed criteria for leakage of PRV into cerebral ventricles. Our data indicate that (i) centrally injected PRV diffuses very little and produces focal injection sites; (ii) PRV is taken up and transported by fibers of passage, although less prominently than found for Ctb; (iii) PRV produces specific and easily identifiable ependymal cell as well as neuronal labeling following ventricular injection. This labeling can be used as a criterion for determining if labeling obtained was due to injected tracer leaking into brain ventricles. In summary, the present study provides new and important information about using PRV to trace central multisynaptic circuitry.