Circadian covariation of norepinephrine and serotonin in the locus coeruleus and dorsal raphe nucleus in the rat

We report robust correlations between concentrations of the neurotransmitters norepinephrine and serotonin in the locus coeruleus and the dorsal raphe nucleus of the brainstem in rats analyzed at 7 different time points over 24 h. We found similar circadian rhythmicities for both monoamines with acrophases just before the onset of the dark period. The monoamine concentrations diminished and the significant intercorrelation between norepinephrine and serotonin in the locus coeruleus disappeared during the night suggestive of a noradrenergic stimulation of dorsal raphe serotonin during the day. Timing of experiments is crucially important in studies on brain monoaminergic indices and their interrelationships.     

The nucleus locus coeruleus modulates local cerebral glucose utilization during noise stress in rats

Local cerebral glucose utilization (LCGU), estimated by the quantitative autoradiographic 2-deoxyglucose technique, was studied in rats with bilateral 6-hydroxydopamine lesions of the locus coeruleus (LC) and in vehicle-injected controls. Unanesthetized animals were studied during exposure to stressful levels of white noise (95 dB) or in relative silence (50 dB). Results indicated that noise caused greater and more widespread increases in LCGU in animals with LC lesions than in vehicle-injected controls. Lesions alone had little or no effect in animals not subjected to noise. Analyses of variance revealed significant treatment interaction effects (intact/lesion x silence/noise) for 37 of 109 regions measured. The pattern of results suggests that the LC acts during stress to limit unnecessary cerebral activity that might interfere with efficient sensory processing and/or the organization of appropriate behavioral responses. In this respect LC function may be similar to those actions of the peripheral sympathetic nervous system that suppress vegetative functions during stress to allow for the performance of coping responses.     

Elevations in nociceptive thresholds following locus coeruleus lesions

Serotonin depletion or lesions of the midbrain dorsal raphe nuclei attenuate both morphine-produced and stimulation-produced analgesia. In contrast, norepinephrine depletion enhances both types of analgesia. To extend these findings, the effects of destruction of the noradrenergic locus coeruleus (LC) upon nociceptive flinch-jump thresholds were investigated. after four preoperative baseline sessions, lesions were placed in the LC bilaterally and nociceptive thresholds were determined for up to five weeks postoperatively. The lesions were localized by monoamine histofluorescence procedures together with conventional histological staining techniques. In 9 of 13 animals, the LC or its ascending dorsal noradrenergic bundle sustained either bilateral or unilateral damage, evidenced by green fluorescent back-up caudal to the lesions. Eight of these animals demonstrated significantly increased jump thresholds. In the remaining four animals, both lesions spared the LC and nociceptive thresholds were either unchanged or significantly decreased. In three of the four, raphe damage was noted, evidenced by yellow fluorescent back-up. The results suggest apparently contrasting roles of norepinephrine and serotonin in nociception.     

The development of sex differences in the locus coeruleus of the rat

Development of sex differences in the locus coeruleus (LC) is investigated. The LC is a sexually dimorphic structure in which the female manifests a larger volume and greater number of neurons than do males. Male and female Wistar rats were sacrificed on prenatal days (E) 16 and 20 and postnatally (P) on days 1, 3, 7, 15, 35, 45, 60, and 90. Male and female rats show a continuous increase in the number of neurons after birth that stops in the males by P45 and in females by P60. These findings point out the existence of different patterns of development in male and female rats and may suggest that sex differences could be established because of the existence of a differential period of neurogenesis in both sexes in the postpubertal period.     

Corticotropin-releasing factor administered into the locus coeruleus, but not the parabrachial nucleus, stimulates norepinephrine release in the prefrontal cortex

Previous studies have indicated that intracerebroventricular application of corticotropin-releasing factor (CRF) activates noradrenergic neurons in the brain stem locus coeruleus (LC) and norepinephrine (NE) metabolism in several brain regions. To assess whether CRF has direct effects on LC noradrenergic neurons, CRF was infused into the LC and concentrations of NE and its metabolites were measured in microdialysates collected from the medial prefrontal cortex (PFM). Infusion of 100 ng of CRF into the LC significantly increased dialysate concentrations of NE and of its catabolite MHPG in the ipsilateral PFM, whereas no significant changes were observed following infusion of artificial CSF. No response was observed when the infusions of CRF occurred outside of the LC, including those in the parabrachial nucleus. Although CRF administered into the LC slightly increased dialysate concentrations of NE in the contralateral PFM, this effect was not statistically significant. The effect of CRF injected into the LC on dialysate NE was prevented by combination with a 10-fold excess of the CRF antagonist, alpha-helical CRF_9–41 indicating some specificity in the response. These results are consistent with anatomical and electrophysiological evidence suggesting that CRF may directly activate noradrenergic neurons in or close to the LC.     

Responses of primate locus coeruleus neurons to simple and complex sensory stimuli

Single and multiple unit recordings were obtained from locus coeruleus (LC) of unanesthetized, chair-restrained monkeys during presentation of a range of sensory stimuli. Tonic activity was higher during alertness or agitation than during behavioral inattentiveness and drowsiness. Low-level, simple auditory stimuli elicited no response, while more intense stimuli evoked phasic discharges in LC activity. The most pronounced responses were elicited by aversive air puffs and by multi-modal naturalistic stimuli such as interactions with the experimenter. The results suggest that sensory stimuli effective in eliciting LC discharge have specific stimulus attributes. It is proposed that the LC is tuned to specifically respond to stimuli which are conspicuous to that species: stimuli which by their physical or behavioral properties evoke a change in the focus of attention. The LC response would thereby contribute to adaptive behavioral responses to such unexpected imperative stimuli. This hypothesis is consistent with earlier suggestions that the LC contributes to behavioral functions such as vigilance and alarm and provides a rigorous framework for future experiments.     

Local opiate withdrawal in locus coeruleus in vivo

Hyperactivity of noradrenergic locus coeruleus (LC) neurons following withdrawal from chronic opiates has been implicated in the opiate withdrawal syndrome. Here, we report that local withdrawal induced in vivo by microinfusion of an opiate antagonist into the LC of morphine-dependent rats marginally, but significantly, activated LC neurons above the level obtained with local naloxone microinfusion in naive rats. This local withdrawal response contributes a significant fraction (∼19%) of the total LC hyperactivity induced by systemic naloxone.     

Regulation of norepinephrine transporter and tyrosine hydroxylase mRNAs after kainic acid-induced seizures

Noradrenergic locus coeruleus (LC) efferents to the forebrain suppress seizures in several models of epilepsy. Using in situ hybridization, we demonstrate that tyrosine hydroxylase (TH) and norepinephrine transporter (NET) but not vesicular monoamine transporter 2 (VMAT2) mRNA levels are transiently elevated in LC neurons following kainic acid-induced status epilepticus. These increases of TH and NET mRNAs and presumably of the proteins themselves might enhance synthesis and reuptake of NE postictally.     

Tyrosine hydroxylase mRNA is increased in old age and norepinephrine uptake transporter mRNA is decreased in middle age in locus coeruleus of Brown-Norway rats

In normal aging, cell loss occurs in the locus coeruleus (LC), the major noradrenergic nucleus in the brain. This study examined changes in the LC of aged rats by measuring mRNA expression for tyrosine hydroxylase (TH) and the norepinephrine uptake transporter (NET). TH and NET mRNA expression were measured by in situ hybridization in young, middle-aged and aged rats. It appears that in middle age, the transporter system responds initially to LC cell loss by decreasing NET mRNA expression. Then, with further aging and cell loss, TH mRNA expression increases which may potentially increase NE synthesis in the remaining neurons. These findings suggest that multiple regulatory components are used to maintain stable noradrenergic synaptic levels despite neuronal loss. Published by Elsevier Science B.V.     

Two physiologically distinct populations of neurons in the ventrolateral medulla innervate the locus coeruleus

Recent anatomic studies indicate that the nucleus paragigantocellularis (PGi), located in the rostral ventrolateral medulla, strongly innervates the locus coeruleus (LC) while no such input derives from the more caudally located lateral reticular nucleus (LRN). In the present study, focal electrical stimulation of the LC was used to antidromically activate neurons in the ventrolateral medulla. A substantial number of PGi neurons were antidromically driven from the ipsilateral LC, while antidromic activation was virtually absent in LRN. Furthermore, several physiologic properties of antidromically driven cells in PGi define two populations within this group of neurons afferent to LC. These findings provide physiologic confirmation of an anatomically identified input to LC.     

Chlorotoxin-mediated disinhibition of noradrenergic locus coeruleus neurons using a conditional transgenic approach

The noradrenergic locus coeruleus (LC) has been implicated in the promotion of arousal, in focused attention and learning, and in the regulation of the sleep/waking cycle. The complex biological functions of the central noradrenergic system have been investigated largely through electrophysiological recordings and neurotoxic lesions of LC neurons. Activation of LC neurons through electrical or chemical stimulation has also led to important insights, although these techniques have limited cellular specificity and short-term effects. Here, we describe a novel method aimed at stimulating the central noradrenergic system in a highly selective manner for prolonged periods of time. This was achieved through the conditional expression of a transgene for chlorotoxin (Cltx) in the LC of adult mice. Chlorotoxin is a component of scorpion venom that partially blocks small conductance chloride channels. In this manner, the influence of GABAergic and glycinergic inhibitory inputs on LC cells is greatly reduced, while their ability to respond to excitatory inputs is unaffected. We demonstrate that the unilateral induction of Cltx expression in the LC is associated with a concomitant ipsilateral increase in the expression of markers of noradrenergic activity in LC neurons. Moreover, LC disinhibition is associated with the ipsilateral induction of the immediate early gene NGFI-A in cortical and subcortical target areas. Unlike previous gain of function approaches, transgenic disinhibition of LC cells is highly selective and persists for at least several weeks. This method represents a powerful new tool to assess the long-term effects of LC activation and is potentially applicable to other neuronal systems.     

Increased extracellular concentrations of norepinephrine in cortex and hippocampus following vagus nerve stimulation in the rat

The vagus nerve is an important source of afferent information about visceral states and it provides input to the locus coeruleus (LC), the major source of norepinephrine (NE) in the brain. It has been suggested that the effects of electrical stimulation of the vagus nerve on learning and memory, mood, seizure suppression, and recovery of function following brain damage are mediated, in part, by the release of brain NE. The hypothesis that left vagus nerve stimulation (VNS) at the cervical level results in increased extracellular NE concentrations in the cortex and hippocampus was tested at four stimulus intensities: 0.0, 0.25, 0.5, and 1.0 mA. Stimulation at 0.0 and 0.25 mA had no effect on NE concentrations, while the 0.5 mA stimulation increased NE concentrations significantly in the hippocampus (23%), but not the cortex. However, 1.0 mA stimulation significantly increased NE concentrations in both the cortex (39%) and hippocampus (28%) bilaterally. The increases in NE were transient and confined to the stimulation periods. VNS did not alter NE concentrations in either structure during the inter-stimulation baseline periods. No differences were observed between NE levels in the initial baseline and the post-stimulation baselines. These findings support the hypothesis that VNS increases extracellular NE concentrations in both the hippocampus and cortex.     

Existence of glutamate in noradrenergic locus coeruleus neurons of rodents

This study distinguished three types of immunolabeled neurons in nucleus locus coeruleus (LC) of the rat and mouse: cells single labeled either for tyrosine hydroxylase-like immunoreactivity (TH-LI) or glutamate (Glu)-LI, and those double labeled for both antigens. Although the double labeled neurons tend to be located in the middle and ventral thirds of the rat LC nucleus, throughout its rostrocaudal extent, such feature was not apparent in the mouse. Quantitatively a majority of neurons cocontaining TH- and Glu-LI were commonly observed in the rat (62%) and mouse (77%) LC. Additional studies utilizing the combined retrograde and immunohistochemical labeling revealed that such a high incidence of coexistence of the TH- and Glu-LI was also represented by coeruleocortical neurons in the rat (69% and 75% of all ipsilateral and contralateral projection cells, respectively). A possible role of coeruleocortical neurons involvement in Glu- and norepinephrine-mediated target neuron dysfunction is discussed.     

Electrophysiological analysis of synaptic transmission between intraocular hippocampus/locus coeruleus co-transplants

We have examined the establishment of functional connectivity between hippocampal and locus coeruleus co-transplants in oculo. Co-transplants were allowed to mature in oculo for 8–54 weeks following grafting and were subsequently removed from the anterior eye chamber for in vitro electrophysiological studies. Single hippocampal transplants in oculo have been shown to exhibit prolonged synaptic responses to local electrical stimulation¹⁷, and similar responses were observed in hippocampal neurons following stimulation of the hippocampal portion of hippocampus-locus coeruleus co-transplants. Electrical stimulation of the locus coeruleus attenuated the afterhyperpolarization in hippocampal neurons elicited by the injection of depolarizing current, an effect that has been described previously in hippocampal slices following direct application of norepinephrine, and this effect was antagonized by pretreatment with the β-adrenergic antagonist timolol. Stimulation of the locus coeruleus also produced both hyperpolarizing and depolarizing changes in the resting membrane potential in hippocampal neurons in 2- and 6-month-old co-transplants. In the 2-month-old co-transplants the responses were primarily depolarizing, and appeared to be mediated by a β-adrenergic receptor, whereas in the 6-month-old co-transplants the responses were more varied. The results suggest that functional α- as well as β-adrenergic receptors develop in oculo, and that the release of norepinephrine at nerve terminals in double grafts produces effects in the hippocampal neurons which are similar to those observed during superfusion of the hippocampal slice preparation with exogenous norepinephrine.     

The effects of peripherald-amphetamine, 4-OH amphetamine, and epinephrine on maintained discharge in the locus coeruleus with reference to the modulation of learning and memory by these substances

d-Amphetamine, 4-OH amphetamine, and epinephrine have been shown in many behavioral studies to facilitate memory when given post-training. The effect of these drugs on the maintained discharge of cells in the locus coeruleus (LC) was investigated using a route of administration (intraperitoneal) and a log-dose range of these drugs comparable to those used in the behavioral experiments.d-Amphetamine profoundly suppressed maintained discharge: an inhibitory effect was observed at every dose (0.1, 1.0, 10.0 mg/kg). In contrast, only the highest dose of 4-OH amphetamine (8.2 mg/kg) inhibited activity in the LC, and this effect was a modest one. Unlike the amphetamines, epinephrine (500 μ/kg) elevated maintained discharge. These results are discussed in the contex of the hypothesized involvement of the LC in the enhancement of memory by these drugs.     

Electrophysiological evidence for locus coeruleus norepinephrine autoreceptor subsensitivity following subchronic administration of -amphetamine

The effect produced by subchronic administration of -amphetamine ( -AMP) on the sensitivity of norepinephrine (NE) autoreceptors in the rat locus coeruleus (LC) was studied by means of single unit recording and microiontophoretic techniques. Twice daily i.p. administration of 5 mg/kg -AMP for one week markedly reduced the ability of i.v. -AMP and microiontophoretic application of clonidine to suppress the firing of LC NE neurons, suggesting strongly that NE autoreceptors became subsensitive. In addition, the firing pattern of NE neurons became ‘disorganized’ following subchronic AMP treatment.     

Hippocampal noradrenergic responses to CRF injected into the locus coeruleus of unanesthetized rats

Intracerebral administration of corticotropin-releasing factor (CRF) activates cerebral noradrenergic neurons. Direct infusion of CRF into the locus coeruleus (LC) increases norepinephrine (NE) release in the cortex and hippocampus as assessed by in vivo microdialysis. In a recent study using in vivo chronoamperometry in anesthetized rats, CRF injected into the LC increased apparent NE release in the hippocampus, but did so after a significant delay, much longer than observed following infusion of glutamate into the same site. Because this delay may have been an artifact of the urethane anesthesia, we developed a method for chronoamperometric recording from the hippocampus of unanesthetized rats. CRF infusion into the LC of such animals induced an increase in the apparent release of hippocampal NE after a mean delay of about 7 min, reached a peak around 16 min after CRF, and dissipated within 30 min. Thus the response closely resembled that previously reported in urethane-anesthetized rats. As in anesthetized rats, glutamate infused into the same site resulted in a much more rapid response (starting within 1 min and with a peak around 7 min). These results suggest that the urethane anesthesia does not substantially alter hippocampal NE release following infusion of CRF into the LC, and that the relatively long delay in the response is not an artifact of the anesthesia. The large differences in the responses to glutamate and CRF suggest that the effects of CRF are not exerted directly on receptors on LC neurons, and more likely reflect indirect actions on other cells in this region.     

Glucocorticoid receptor-immunoreactivity in corticotrophin-releasing factor afferents to the locus coeruleus

The stress-related neurohormone, corticotropin-releasing factor (CRF), also acts as a neurotransmitter to activate the brain noradrenergic nucleus, locus coeruleus (LC). Previous electrophysiological findings demonstrating that tonic CRF secretion in the LC is increased in adrenalectomized rats suggest that activity of certain CRF afferents to the LC is under inhibitory regulation by endogenous corticosteroids. The present study was designed to identify putative CRF afferents to the LC that may be regulated by glucocorticoids. Retrograde tract tracing from the rat LC and pericoerulear regions was combined with immunohistochemistry to visualize CRF and glucocorticoid receptors in the same sections of rat brain. The retrograde tracer, wheat germ agglutinin conjugated to horseradish peroxidase coupled to gold (WGA–Au–HRP) was injected into either the nucleus LC or the rostrolateral pericoerulear region (peri-LC), where CRF-immunoreactive terminals have been demonstrated to synapse with LC dendrites. Sections were processed to visualize the tracer, as well as CRF- and glucocorticoid receptor-immunoreactivity. Following injections of WGA–Au–HRP into the nuclear LC, triple labeled neurons were observed primarily in Barrington's nucleus, where 74±4% of retrogradely labeled CRF-immunoreactive neurons colocalized glucocorticoid receptor immunoreactivity. In contrast, injections that incorporated the rostrolateral peri-LC retrogradely labeled numerous neurons that were immunoreactive for both CRF and glucocorticoid receptors in the central nucleus of the amygdala. Thus, 94±2% of the retrogradely labeled CRF-immunoreactive neurons in the central nucleus of the amygdala colocalized glucocorticoid receptor immunoreactivity. Additionally, triple labeled neurons were observed in the bed nucleus of the stria terminalis following WGA–Au–HRP injections that incorporated the rostrolateral peri-LC. The present results implicate Barrington's nucleus, the central nucleus of the amygdala and the bed nucleus of the stria terminalis as glucocorticoid-sensitive sources of CRF that can influence the LC-noradrenergic system. Alterations in glucocorticoid levels or glucocorticoid receptor function would be predicted to affect the impact of these specific CRF systems on LC activity.     

Stress, antidepressant drugs, and the locus coeruleus

This review presents a synthesis of a large body of seemingly inconsistent literature on the role of the locus coeruleus-norepinephrine (LC-NE) system and the corticotropin-releasing hormone (CRH)-median eminence system in mediating the CNS effects of stress and the therapeutic effects of antidepressant drugs. The clinical implications of these findings for the etiology and treatment of stress-related psychiatric disorders such as depression will be discussed.     

The brain norepinephrine system,stress and cardiovascular vulnerability

Chronic exposure to psychosocial stress has adverse effects on cardiovascular health, however the stress-sensitive neurocircuitry involved remains to be elucidated. The anatomical and physiological characteristics of the locus coeruleus (LC)-norepinephrine (NE) system position it to contribute to stress-induced cardiovascular disease. This review focuses on cardiovascular dysfunction produced by social stress and a major theme highlighted is that differences in coping strategy determine individual differences in social stress-induced cardiovascular vulnerability. The establishment of different coping strategies and cardiovascular vulnerability during repeated social stress has recently been shown to parallel a unique plasticity in LC afferent regulation, resulting in either excitatory or inhibitory input to the LC. This contrasting regulation of the LC would translate to differences in cardiovascular regulation and may serve as the basis for individual differences in the cardiopathological consequences of social stress. The advances described suggest new directions for developing treatments and/or strategies for decreasing stress-induced cardiovascular vulnerability.