Alterations of norepinephrine metabolism in rat locus coeruleus neurons in response to axonal injury

Adult male Sprague-Dawley rats were injected in the right cerebral hemisphere with the neurotoxin 6-hydroxydopamine (6-OHDA) at a site which interrupted the noradrenergic axons ascending from the locus coeruleus (LC). Distal to the injection site ('posterior cortex'), levels of norepinephrine (NE), dopamine-beta-hydroxylase (D beta H) and tyrosine hydroxylase (TH) fell to 39-42% of control levels ipsilateral to the lesion over the first 25 days, while contralateral levels fell to 32-73% of control during this time. These changes were paralleled by a 63% decrease in the high affinity uptake of [3H]NE in the ipsilateral posterior cortex at 12 days after the lesion. Both ipsilateral and contralateral levels of NE and D beta H fell in the LC during this time, while LC TH showed variable increases and decreases in activity. At 3 months after right cortical 6-OHDA injections, posterior cortical levels of NE, D beta H and TH, as well as the high affinity uptake of [3H]NE, had returned to control levels suggesting that some type of regeneration or axonal sprouting had occurred. Axonal transport of D beta H and TH was assessed by measuring the accumulation of enzyme activity proximal to a 6-OHDA lesion made in the more caudal portion of these same LC axons. Transport of D beta H fell to 7-40% of control from 2 to 24 days and rose to 160% of control by 3 months after the lesion. TH transport was decreased to only 61% of control only at 24 days and returned to control levels by 3 months. These studies document that there is independent regulation of the metabolism of the NE synthetic enzymes, D beta H and TH, during the degeneration and subsequent regeneration or collateral sprouting of injured distal axons of LC noradrenergic neurons.     

The action of norepinephrine in the rat hippocampus. III. Hippocampal cellular responses to locus coeruleus stimulation in the awake rat.

The behavioral and physiological effects of electrical stimulation of the nucleus locus coeruleus (LC) were studied in the awake rat. LC electrodes consistently supported high rates of self-stimulation (SS). LC stimulation also inhibited spontaneous cellular discharges in the hippocampus (HPC). Both the LC-induced inhibition of HPC units and the LC-evoked SS behavior were antagonized by alpha-methyltyrosine and 6-hydroxydopamine. In addition, chlorpromazine and diethyldithiocarbamate antagonized LC-induced inhibition of HPC units. D-Amphetamine facilitated SS behavior and reduced spontaneous HPC unit activity. The reinforcing properties of LC stimulation correlate closely with inhibition of cellular activity in the hippocampus; both actions appear to be mediated by norepinephrine.     

Target cell stimulation and inhibition of norepinephrine uptake in dissociated rat locus coeruleus cultures

Primary cultures from dissociated locus coeruleus (LC) neurons of 14-day-old (E14) fetal rats were grown in vitro in serum complemented medium. Noradrenergic cells were identified using immunocytochemical staining for tyrosine hydroxylase (TH) antibody. Maturation of noradrenergic neurons was assessed by measuring the high-affinity uptake of [3H]norepinephrine (NE). The presence of hippocampal cells stimulated the specific uptake of [3H]NE by LC cells only when plated at low density. Increasing the concentration of hippocampal cells resulted in a 50% decrease in NE uptake by LC cells. A similar inhibitory effect was observed with striatal cells. The inhibition exerted by striatal cells appears to be developmentally regulated, with E18 exerting a stronger inhibitory effect than E15 striatum. The decrease in [3H]NE uptake in hippocampal-LC cocultures was due to a decrease in uptake by individual noradrenergic neurons. For a given plating density, the decrease in uptake of [3H]NE per noradrenergic cell in LC culture was only half the decrease in the cocultures, suggesting a target-associated effect rather than density-derived toxic effect. In culture conditions which favored neuronal but not glial survival, the stimulatory target effect was evident, and the inhibitory effect was absent. Medium conditioned by target glial cells had a marked stimulatory effect on [3H]NE uptake. Glial feader-layer had a strong inhibitory effect on [3H]NE uptake in serum-containing medium. We suggest that both neurons and glia mediate the target-stimulatory effect, whereas the inhibitory effect is mediated by direct contact between target glia and LC neurons.     

Discrete local application of corticotropin-releasing factor increases locus coeruleus discharge and extracellular norepinephrine in rat hippocampus.

The most prominent afferents impinging upon the noradrenergic neurons of the locus coeruleus (LC) utilize GABA and glutamate. However, peptide neurotransmitters such as galanin, neuropeptide Y, and corticotropin-releasing factor (CRF) have also been localized to LC afferents. The evidence for CRF modulation of LC activity was examined in the present studies. Specifically, the impact of local CRF administration on both LC-NE discharge characteristics and release of norepinephrine (NE) in hippocampus was determined. First, the ability of CRF microinfused into the LC area to increase NE efflux in the dorsal hippocampus was determined using in vivo microdialysis techniques in awake rats. CRF into the LC dose-dependently increased extracellular NE in the ipsilateral hippocampus. Second, a more detailed analysis was performed in halothane-anesthetized rats by characterizing the electrophysiological activity of LC-NE neurons in response to local application of CRF. Changes in the firing rate and pattern of single LC-NE neurons were measured while simultaneously monitoring the extracellular level of NE in hippocampus. A dose of 30 ng CRF applied directly into LC via pressure ejection elicited an 88% increase in the discharge rate of LC-NE neurons and increased the incidence of burst firing from 14% to 33%. This manipulation simultaneously increased extracellular NE in hippocampus by 63%. The CRF-induced increases in discharge rate of LC-NE neurons and extracellular NE efflux in hippocampus were prevented by prior i.c.v. administration of the CRF antagonist, d-PheCRF(12-41 )(3 microg / 3 microl). The present findings demonstrate that CRF applied directly into the LC increases both the activity of LC-NE neurons and the release of NE in an LC terminal region. The shift in activity of LC-NE neurons to more burst-like firing in response to CRF may provide a means for enhanced release of NE in LC projection fields. This is the first report to demonstrate a dose-dependent increase in extracellular NE levels evoked by intra-LC infusion of CRF in unanesthetized animals.     

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 locus coeruleus, norepinephrine, and memory in newborns

Use of learned odor cues by newborn rats is critical for pup survival. Rat pups acquire approach responses to maternal odors through an associative conditioning mechanism. This learned behavioral response is accompanied by a modification of olfactory bulb neural response patterns to the learned odor. Both the behavioral and neural reponse changes involved and require norepinephrine release in the olfactory bulb. The source of this norepinephrine is the locus coeruleus. It is proposed that the unique response properties of the locus coeruleus during the early postnatal period in the rat may facilitate acquisition of these critical early memories.     

Facilitation of lumbar monosynaptic reflexes by locus coeruleus in the rat

The present study was initiated to delineate whether species difference exists between cats and rats in the descending influence of locus coeruleus (LC) on spinal motoneuronal activity. In male Sprague-Dawley rats anesthetized with chloral hydrate (400 mg/kg, i.p.), localized activation of LC promoted an exclusive facilitation of lumbar spinal extensor and flexor monosynaptic reflexes (MSRs). Such LC-evoked potentiations may vary in degree (37.5-147.4%), duration (70.6-72.9 ms) and latency (3.0-5.5 ms) among different animals. While minimally affecting the control MSRs, the alpha 1-adrenoceptor blocker prazosin (20 micrograms/kg, i.v.) significantly antagonized the enhancing effect of the LC on MSRs, suggesting the participation of noradrenergic neurotransmission in the process. Since these results are in general agreement with previous observations from our laboratory on the cat, we conclude that the LC exerts similar facilitatory actions on both extensor and flexor motoneuron activity of the hindlimb in at least two animal species, rat and cat.     

Anomalous rectification in rat locus coeruleus neurons

Rat locus coeruleus neurons recorded under current clamp conditions show anomalous rectification (AR) as indicated by a progressive decrease in slope resistance measured with hyperpolarizing current pulses of increasing amplitude. AR was most prominent at potentials more negative than the K+ equilibrium potential. AR was strongly dependent on external K+ concentration and was blocked by external Ba2+ and Cs+, and intracellular injection of acetate.     

Ultrastructural changes in rat locus coeruleus induced by chronic opioids

The locus coeruleus (LC) is a major noradrenergic nucleus in the brain. The activity of the LC neurons is chronically regulated by opioids. So far, very little is known about the morphological changes induced by chronic treatment with opioids. In the present study, the effects of chronic treatment with morphine and dihydroetorphine, a new narcotic analgesic with lower physical dependence potential than morphine, were investigated on the ultrastructure of the rat LC. Rats received saline or increasing doses of morphine or dihydroetorphine for 5 days by twice daily subcutaneous injections. Withdrawal was precipitated in half of the opioid-treated rats by a single intraperitoneal injection of naloxone 4 h after the last injections of opioids. The ultrastructure of the LC was examined by electron microscopy. Results showed that chronic morphine treatment induced a marked injury to the LC neurons. The primary changes in the cell body were the indentation of nuclei, the fragmentation and degranulation of rough endoplasmic reticulum, as well as the disaggregation of polyribosomes. Myelinoid bodies were seen in the processes. An accumulation of presynaptic vesicles was observed in some of the terminals which formed synaptic junctions with the LC neurons as compared to the normal controls. Naloxone-precipitated withdrawal from morphine did not stop the morphine-induced injury on the LC neurons except that less accumulation of presynaptic vesicles occurred. Chronic dihydroetorphine treatment only induced a slight change in the ultrastructure of the LC neurons. These results indicate that the LC neurons are more vulnerable to chronic treatment with morphine than to that with dihydroetorphine. Received: 30 November 1995 / Revised: 11 January 1996 / Revised, accepted: 3 February 1997     

Cardiovascular alteration by nucleus locus coeruleus in spontaneously hypertensive rat

Biphasic electrical stimulation of the nucleus locus coeruleus (LC) produced frequency-related pressor responses associated with increased heart rate in normotensive rats (WKY) at both 50 and 100 μA. In contrast, spontaneously hypertensive rats (SHR) demonstrated small depressor responses at 50 μA and 50 Hz, but no consistent pressure or heart rate changes at either 50 or 100 μA stimulation. The LC pressor threshold current (current necessary to increase arterial pressure by ≥ 10 mm Hg) was higher in SHR than WKY (136 ± 5 μA vs.84 ± 7 μA; P < 0.001); and only the SHR LC demonstrated a depressor threshold (current necessary to reduce arterial pressure by ≥ 10 mm Hg) (53 ± 3 μA). These threshold current levels did not seem to change with acute alterations of arterial pressure produced by either bilateral carotid artery occlusion or carotid sinus stimulation. Therefore, these studies indicate altered levels of responsiveness of the SHR LC to stimulation but that the pressor function of the LC does not seem to be importantly involved in further elevation of abnormally high arterial pressure in the SHR.     

Functional reinnervation of rat hippocampus by locus coeruleus implants

Transplants of the embryonic locus coeruleus (LC) region were implanted into the circuity of the hippocampal formation in adult rats in which the normal adrenergic afferents to the hippocampus had been removed. The growth of new adrenergic axons from the implant in the denervated hippocampus was followed for 1–14 months after surgery by means of fluorescence histochemistry, and the function of the implant-hippocampal connections was tested electrophysiologically after 2–3 months survival. In the successful cases the entire hippocampal formation was reinnervated from the LC implant within 3–6 months after operation, and the newly formed innervation still persisted unchanged by 14 months. The reinnervation was equally effective irrespective of the route by which the axons entered the hippocampus, i.e. along the lesioned fornix-fimbria or along a retrosplenial route. The pattern formed by the ingrowing LC axons mimicked to a large extent that of the normal LC afferents. Little growth was seen into denervated terminal fields of the commissural, septal or entorhinal afferents, pointing to a preference of the ingrowing LC fibers for the areas normally innervated by adrenergic afferents. In the electrophysiological experiments, stimulation of the LC implants caused (in 20 out of 29 cells monitored) an inhibition of the spontaneous activity of neurons in the host hippocampus. This inhibition had a relatively long latency and a long duration, similar to that observed after stimulation of the innate LC in the intact rat. As in the normal rat, the inhibitory responses were blocked by systemic or local application of the beta-adrenergic receptor blockers propranolol or sotalol.

It is concluded that the adult rat brain is capable of carrying out all steps involved in correct functional reinnervation of a denervated region. Moreover, the implant-hippocampal preparation should be a highly suitable model system for functional studies of a central noradrenergic connection.     

Raphe projections to the locus coeruleus in the rat

Afferent projections to the locus coeruleus from the various raphe nuclei, particularly of the midbrain (nuclei raphe dorsalis and medianus) and pons (nuclei raphe pontis and magnus), have been studied in the rat by retrograde transport methods using horseradish peroxidase (HRP). The locus coeruleus, in both its dorsomedial and ventrolateral divisions, and in its various anterior-posterior components, were injected with 0.05 μl of horseradish peroxidase following which various structures of the brainstem, particularly the raphe nuclei, were examined for HRP reactive cells. It was found that injections in most components of the locus coeruleus were associated with HRP positive cells in varying degrees of density in the nuclei raphe dorsalis, medianus, pontis, and magnus, with considerably sparser labelling in the anterior aspects of the medullary raphe nuclei pallidus and obscurus. Labelled cells were also seen in the nuclei of the solitary tract, contralateral locus coeruleus, lateral reticular areas of the pons and midbrain, nuclei pontis oralis and caudalis, vestibular nuclei, mesencephalic nucleus of the trigeminal nerve, fastigial nuclei of cerebellum and medial parabrachial nuclei. These data, showing widespread innervation of the locus coeruleus from all raphe nuclei, as well as many other brainstem areas, in the rat support the general view of heavy innervation of the locus coeruleus from both extra-raphe and raphe nulcei. These latter raphe projections, probably serotonergic in nature, provide anatomical support for the various experiments indicating considerable regulation of locus coeruleus activities, such as phasic events of REM sleep, among others, by most of the raphe nuclei. Thus, various activities of the locus coeruleus could be modulated or regulated by widespread projections from most raphe nuclei as well as several other regions of the brainstem.     

The action of norepinephrine in the rat hippocampus. IV. The effects of locus coeruleus stimulation on evoked hippocampal unit activity.

Activity of neurons in the hippocampus (HPC) was recorded in awake, freely moving rats. Most cells were inhibited by either a loud auditory stimulus (tone) or by electrical stimulation of the nucleus locus coeruleus (LC). The inhibitory responses to the tone were antagonized by drugs that interfere with central noradrenergic transmission. When LC stimulation was used as the unconditioned stimulus in a classical conditioning paradigm, previously inhibitory responses to the tone were reinstituted. When behaviorally subthreshold LC stimulation preceded a tone which was correlated with food, the existing conditioned response to the tone was potentiated. These data suggest that the generalized inhibitory response of HPC neurons to a tone is modulated by the noradrenergic pathway and that experimental activation of LC can potentiate HPC responses to behaviorally significant conditioned stimuli.     

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

The effect produced by subchronic administration of D-amphetamine (D-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 D-AMP for one week markedly reduced the ability of i.v. D-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.     

Corticotropin-releasing factor in the norepinephrine nucleus, locus coeruleus, facilitates behavioral flexibility

Corticotropin-releasing factor (CRF), the stress-related neuropeptide, acts as a neurotransmitter in the brain norepinephrine nucleus, locus coeruleus (LC), to activate this system during stress. CRF shifts the mode of LC discharge from a phasic to a high tonic state that is thought to promote behavioral flexibility. To investigate this, the effects of CRF administered either intracerebroventricularly (30-300?ng, i.c.v.) or directly into the LC (intra-LC; 2-20?ng) were examined in a rat model of attentional set shifting. CRF differentially affected components of the task depending on dose and route of administration. Intracerebroventricular CRF impaired intradimensional set shifting, reversal learning, and extradimensional set shifting (EDS) at different doses. In contrast, intra-LC CRF did not impair any aspect of the task. The highest dose of CRF (20?ng) facilitated reversal learning and the lowest dose (2?ng) improved EDS. The dose-response relationship for CRF on EDS performance resembled an inverted U-shaped curve with the highest dose having no effect. Intra-LC CRF also elicited c-fos expression in prefrontal cortical neurons with an inverted U-shaped dose-response relationship. The number of c-fos profiles was positively correlated with EDS performance. Given that CRF excites LC neurons, the ability of intra-LC CRF to activate prefrontal cortical neurons and facilitate EDS is consistent with findings implicating LC-norepinephrine projections to medial prefrontal cortex in this process. Importantly, the results suggest that CRF release in the LC during stress facilitates shifting of attention between diverse stimuli in a dynamic environment so that the organism can adapt an optimal strategy for coping with the challenge.     

Regulation of G proteins by chronic morphine in the rat locus coeruleus

A possible role for G proteins in contributing to the chronic actions of opiates was investigated in the rat locus coeruleus (LC). The LC is a relatively homogeneous brain region that appears to play an important role in mediating acute and chronic opiate action in animals, as well as in humans. It was found that chronic, but not acute, treatment of rats with morphine, under conditions known to induce states of opiate tolerance and dependence, produced an increase in the level of pertussis toxin-mediated ADP-ribosylation of G proteins in the LC. The morphine-induced increase in ADP-ribosylation occurred in both Gi and Go, and was observed over a 30-fold range of NAD concentrations used. Concomitant treatment of rats with the opiate receptor antagonist naltrexone blocked the ability of morphine to produce this effect. In contrast, chronic morphine had no effect on pertussis toxin-mediated ADP-ribosylation of Gi and Go in the other brain regions studied, including the neostriatum, frontal cortex, and dorsal raphe. Chronic morphine also had no effect on cholera toxin-mediated ADP-ribosylation of Gs in the LC and these other brain regions. Preliminary immunoblot analysis revealed that increased ADP-ribosylation levels of the alpha subunit of Go in the LC were associated with equivalent increases in the immunoreactivity of this protein in this brain region. It is possible that the observed regulation of G-proteins by morphine in the LC represents part of the changes that underlie opiate addiction in these neurons.     

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.     

Tyrosine hydroxylase and norepinephrine transporter mRNA expression in the locus coeruleus in Alzheimer's disease

Despite the loss of locus coeruleus (LC) noradrenergic neurons in Alzheimer's disease (AD), cerebrospinal fluid norepinephrine (NE) levels are normal or increased in AD. This paradox suggests compensatory upregulation of NE synthetic capacity or downregulation of the NE transporter (NET) in the remaining LC neurons. LC tyrosine hydroxylase (TH) mRNA expression in the LC was measured in AD subjects (n=5) and in age and gender comparable non-demented subjects (n=6). When AD subjects were divided into those still ambulatory prior to death (CDR 3/4) and those in a prolonged 'vegetative' state prior to death (CDR 5), differences among groups became apparent at specific levels of the LC. In CDR 3/4 AD subjects there was increased TH mRNA expression per neuron compared to non-demented subjects in the caudal half of the LC. However, expression of NET mRNA in the same subjects was not significantly different at any level of the LC. These preliminary results suggest an upregulation of NE biosynthetic capacity in at least some LC neurons in AD prior to the very late stage of the disease.     

Transient homologous mu-opioid receptor desensitization in rat locus coeruleus neurons.

Opioid agonists hyperpolarize neurons of the locus coeruleus (LC) in the slice preparation. When opioids were applied at concentrations that caused a maximum hyperpolarization, the membrane potential hyperpolarized to a peak (about 30 mV) in the first minute and then declined over a period of 5 min. In addition, following the washout, the amplitude of the hyperpolarization induced by a lower concentration of opioid was significantly reduced as compared to control. The original response to both the low and the high concentrations of opioid recovered after removal of opioids for about 20 min. The decline in response, termed "acute desensitization," was observed only with concentrations of opioids that caused a maximum hyperpolarization and was dependent on the concentration of opioid applied (EC50 for [Met5]-enkephalin (ME), between 3 and 5 microM). The response to ME (300 nM) was reduced to 6% of control following washout of a 5-min application of ME (30 microM), whereas the response to noradrenaline (300 nM) was reduced to 75% of control. The acute desensitization therefore was selective for the opioid receptor with marginal cross-desensitization to the alpha 2-adrenoceptor-mediated hyperpolarization. The desensitization still occurred following treatment with beta-chlornaltrexamine (beta-CNA), to decrease receptor reserve, as well as in cells taken from animals treated chronically with morphine. The mechanism for the acute desensitization was investigated using agents thought to alter kinase activity. This acute desensitization may represent an initial stage in the development of tolerance produced by chronic administration of opioids.