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Effects of local MAO inhibition in the locus coeruleus on extracellular serotonin and 5-HIAA during exposure to sensory and cardiovascular stimuli.

Previously, we have shown that in the presence of pargyline the release of serotonin (5-HT) in the locus coeruleus is modulated by various sensory stimuli and blood pressure fluctuations. The aim of the present study was to investigate whether local inhibition of monoamine oxidase (MAO) influences basal and stimulus-induced release of 5-HT in the locus coeruleus. For this purpose, the locus coeruleus was superfused in the absence and in the presence of the MAO inhibitor pargyline. Additionally, we examined whether the release of the 5-HT metabolite 5-hydroxy-indole acetic acid (5-HIAA) in the locus coeruleus is altered in response to stimuli. The locus coeruleus of the conscious rat was superfused through a push-pull cannula with artificial cerebrospinal fluid (CSF). 5-HT and 5-HIAA were determined in the superfusate. The basal release rate of 5-HT and the basal outflow of 5-HIAA averaged 2.0 fmol/min and 69 fmol/min, respectively. The basal release rate of 5-HT and the 5-HIAA outflow were tetrodotoxin (TTX)-sensitive. In the absence of pargyline, the sensory stimuli noise stress or tail pinch, applied for 10 min, increased 5-HT and 5-HIAA outflow by 50-70%. In contrast, an experimentally induced rise in blood pressure for 10 min enhanced 5-HT release by 50%, but had no effect on 5-HIAA outflow. The release of 5-HT and/or 5-HIAA elicited by sensory stimuli or a blood pressure rise was abolished by TTX. Addition of pargyline to the CSF enhanced 5-HT release fourfold and slightly decreased 5-HIAA outflow. These levels remained stable throughout the entire observation period of 8 h. In the presence of pargyline, 5-HT release elicited by noise, tail pinch and increase in blood pressure was enhanced. It is concluded that superfusion with pargyline enhances 5-HT release and reduces 5-HIAA outflow in the locus coeruleus. Furthermore, the ability of sensory stimuli and baroreceptor activation to enhance 5-HT release is preserved during a prolonged pargyline-induced increase in extracellular 5-HT. Since sensory stimuli enhanced, while baroreceptor activation did not influence 5-HIAA outflow, 5-HIAA is not a reliable index for short-term changes in the activity of serotonergic neurons in the locus coeruleus.

Acoustic Stimulation↗

Decrease in the reactivity of locus coeruleus neurons to hypotension after an increase in their tyrosine hydroxylase content: a subregional in vivo voltammetry study in the rat.

The aim of the present work was to determine if noradrenergic neurons of the anterior and the posterior subregions of the locus coeruleus exhibit a difference in reactivity in response to sodium nitroprusside-induced arterial hypotension, and if the pharmacological induction of tyrosine hydroxylase by RU24722 modifies the reactivity of locus coeruleus neurons to this hypotensive stimulus. Previous findings have demonstrated that administration of RU24722 increases the concentration of tyrosine hydroxylase in the rat locus coeruleus by two different mechanisms in the anterior and in the posterior locus coeruleus subregions. The goal of the present study was to measure in vivo the changes in catecholaminergic metabolism in the locus coeruleus after treatment with RU24722 using differential normal pulse voltammetry (DNPV). In vehicle-treated rats, arterial hypotension increased catecholaminergic metabolism with the same pattern in the two locus coeruleus subregions. However, the changes in the magnitude of the catechol oxidation current throughout the recording period were significantly smaller in the posterior subregion (P < 0.001). In the RU24722-pretreated rats, there was a 39% increase in tyrosine hydroxylase and dihydroxyphenylacetic acid in the locus coeruleus. The functional reactivity to hypotension measured by DNPV was significantly decreased (P < 0.001) in both the anterior and posterior locus coeruleus subregions with RU24722 treatment. Therefore, this study suggests that the response of locus coeruleus cells to a hypotensive stimulus depends upon the intracellular tyrosine hydroxylase concentration both in the basal condition and during pharmacological induction of tyrosine hydroxylase gene expression.

3,4-Dihydroxyphenylacetic Acid↗

Contraction of urinary bladder by central norepinephrine originating in the locus coeruleus.

Studies were performed to elucidate the role of the locus coeruleus, which is rich in norepinephrine-containing cell bodies, in vesical function using alpha-chloralose anesthetized cats. Stimulation of the locus coeruleus caused contraction of the urinary bladder, which was not affected by transection of the bilateral hypogastric nerves, but blocked by intravenous administration of hexamethonium, a ganglion blocking agent. In animals with transected hypogastric nerves, the locus coeruleus-induced contraction was inhibited by intrathecal administration of phentolamine (alpha-blocker) and prazosin (alpha 1-blocker), but not affected by intrathecal sotalol (beta-blocker) or yohimbine (alpha 2-blocker). In animals treated with reserpine, the locus coeruleus-induced contraction was enhanced by intravenous application of L-dopa, a precursor of norepinephrine. These results suggest that norepinephrine derived from the locus coeruleus activated preganglionic neurons in the sacral intermediolateral nuclei via alpha 1-receptors, thereby producing urinary bladder contraction.

Animals↗

Unilateral, but not bilateral, locus coeruleus lesions facilitate recovery from sensorimotor cortex injury.

This study investigates the role of the locus coeruleus in recovery from sensorimotor cortex injury. Unilateral locus coeruleus lesions given 2 weeks prior to unilateral sensorimotor cortex injury facilitate subsequent motor recovery compared to animals with only a sensorimotor cortex injury, while bilateral locus coeruleus lesions severely retard motor recovery. The results suggest that recovery of function from the cortical injury is facilitated as long as a sufficient amount of the noradrenergic system remains intact, perhaps to provide a basis for compensatory sprouting. The results also suggest that recovery does occur in the absence of the locus coeruleus, indicating that the noradrenergic system is not necessary for recovery to occur after the cortical injury.

Animals↗

Divergent axon collaterals of rat locus coeruleus neurons: demonstration by a fluorescent double labeling technique.

The distribution of the noradrenaline-containing neurons of the rat locus coeruleus has been investigated with retrograde labeling techniques using two different fluorescent tracers. Injections were placed in the prefrontal cortex, the striatum, the thalamus, the hippocampus, the cerebellar cortex and the lumbar spinal cord. No evidence for locus coeruleus projections to the striatum was found. Injections in the cortex, thalamus and hippocampus revealed not only ipsilateral but also contralateral labeling of cells in the locus coeruleus. Following unilateral or bilateral homo- or heterotopic injections of the two tracers several cells of the locus coeruleus were double labeled. Combined injections of the two fluorophores in any of these forebrain areas and in the spinal cord also produced double labeled cells. The majority of double labeled cells was located in an area between the ventral and the dorsal parts of the locus coeruleus. These results indicate that individual neurons of the locus coeruleus have the possibility to influence adrenergic receptors at remote areas in the central nervous system.

Animals↗

Cell suspension grafts of noradrenergic locus coeruleus neurons in rat hippocampus and spinal cord: reinnervation and transmitter turnover.

Fetal noradrenergic neurons from the brain stem locus coeruleus region can be successfully grafted as a dissociated cell suspension provided that the dissociation is done in the absence of any trypsin digestion step. The survival, fiber outgrowth and biochemical function of locus coeruleus neurons, taken from 13- to 15-day-old rat embryos, have been studied after injection into the dorsal hippocampal formation and the thoracolumbar spinal cord in adult rats. All rats were treated with an i.v. injection of 6-hydroxydopamine prior to grafting to remove the intrinsic locus coeruleus projections to these areas, and they were taken for fluorescence histochemical or biochemical analyses 2-7 months after transplantation. Up to 330 surviving noradrenaline neurons were found at each implantation site (injected with 2-3 microliters of cell suspension) which represents an estimated survival rate of about 40%. In the most successful cases the entire dorsal hippocampal formation, and an approximately 4 cm long segment of the thoracolumbar spinal cord, was supplied with a new noradrenaline-containing terminal network, which reached normal densities in the regions closest to the grafts. In the hippocampal formation, in particular, the ingrowing axons re-established a laminar innervation pattern which resembled that of the normal locus coeruleus afferents. In the hippocampus, two 2-microliters injections of locus coeruleus cell suspension restored the total hippocampal noradrenaline content to an average of 55%, and the noradrenaline synthesis rate (as assessed by the rate of DOPA accumulation after synthesis inhibition) was found to be close to normal in the graft-reinnervated specimens. In the spinal cord, two 3-microliters injections restored the noradrenaline level in the thoracolumbar cord (a 4.5 cm long segment) to an average of 22% of normal, with the highest individual levels being close to normal. Determinations of the noradrenaline metabolite 3,4-dihydroxy-phenylethyleneglycol indicated that the rate of noradrenaline metabolism in the graft-reinnervated spinal cord was close to that of the normal intact spinal cord. The results demonstrate the potential of the suspension grafting technique for extensive noradrenergic reinnervation of the hippocampal formation or large portions of the spinal cord. Fetal locus coeruleus neurons implanted in this way can re-establish fairly normal terminal innervation patterns and reinstate noradrenaline turnover and metabolism in a previously denervated central target.

Adrenergic Fibers↗

Ubiquitin expression in globose tangles in the locus coeruleus in brains of patients with Alzheimer's and Parkinson's diseases.

The locus coeruleus belongs to brain areas exhibiting remarkable neuronal loss already during physiological aging and very early neurofibrillary tangles. We performed a semiquantitative, immunocytochemical study focused on the expression of ubiquitin in neurofibrillary tangles in the locus coeruleus as compared with the central superior nucleus. The locus coeruleus exhibited later ubiquitin expression in the neurofibrillary tangles, a lower percentage of ubiquitinated tangles and more frequently granular cytoplasmic staining with Tau-1 than the central superior nucleus. These results suggesting different, probably delayed, processing of Tau protein in the locus coeruleus might contribute additionally to cell injury in this area.

Aged↗

Cortical effects of the electrical stimulation of the locus coeruleus.

The results of the electrical stimulation of the locus coeruleus in curarized cats, and the effects upon the observed cortical responses of 5,6-dihydroxytryptamine or 6-hydroxydopamine injected intracisternally or applied in the subarachnoideal space were studied. The changes were followed on the strychnine spikes produced after the topical application of this drug. Stimulation of the locus coeruleus elicited a response in every area of the cortex explored. The response was complex, showed a phase of depression and a phase of enhancement both mixed in different proportions. The depressant reaction was more marked with lower strengths of stimulation and the enhancement reactions appeared more evident with the stronger stimuli. Both these reactions were blocked by the previous administration of 6-hydroxydopamine and only the depressant one was practically abolished by 5,6-dihydroxytryptamine. It is concluded that a complex interaction exists between the noradrenergic neurons of the locus coeruleus and the serotonergic neurons, probably those of the raphe nuclei. It is suggested that the axons of the noradrenergic neurons in their way to the cortex, give off collaterals that innervate some serotonergic neurons. The monoamines liberated by these axon terminals at the cerebral cortex would act as neurohumoral agents.

Animals↗

The release of catecholamines in hypothalamus and locus coeruleus is modulated by peripheral chemoreceptors.

To investigate whether impulses from chemoreceptors influence the release of catecholamines in the hypothalamus and the locus coeruleus, the two brain areas were superfused simultaneously and bilaterally with artificial cerebrospinal fluid through push-pull cannulae. The release of catecholamines was determined in the superfusate before and during chemoreceptor stimulation by bicarbonate solution saturated with carbon dioxide (CO2-NaHCO3) or KCN. Experiments were carried out on intact cats after carotid body denervation (CD). Intracarotid infusion of CO2-NaHCO3 increased arterial blood pressure and enhanced the release of noradrenaline but not dopamine in the posterior hypothalamus and the locus coeruleus. Following CD, the enhancing effect of CO2-NaHCO3 on the noradrenaline release in the posterior hypothalamus was abolished, while the effect on blood pressure was slightly enhanced. CD reversed the NaHCO3-induced release of noradrenaline in the locus coeruleus to a decreased noradrenaline outflow. Intracarotid infusion of KCN led to a fall in blood pressure. KCN increased the release rates of noradrenaline and, to a lesser extent, that of dopamine in the posterior hypothalamus, as well as the release of noradrenaline in the locus coeruleus. CD abolished the KCN-induced fall of blood pressure and the increased release of noradrenaline and dopamine in the posterior hypothalamus. Similar to CO2-NaHCO3, the enhancing effect of KCN on the noradrenaline release in the locus coeruleus was reversed following CD to a reduced noradrenaline outflow. Superfusion of the posterior hypothalamus and the locus coeruleus with KCN did not influence either blood pressure or the release rates of noradrenaline and dopamine in these brain areas. The findings show that impulses originating from chemoreceptors of the carotid body increase the release rates of the catecholamines in the posterior hypothalamus and the locus coeruleus, thus underlining the importance of catecholaminergic neurons of these brain areas in cardiovascular control.

Animals↗

Comparative study of the effects of desipramine and reboxetine on locus coeruleus neurons in rat brain slices.

Several studies have suggested that the locus coeruleus may play an important role in the pathophysiology of depression. The aim of this study was to characterize, using single-unit extracellular recordings, the in vitro effects of the noradrenaline reuptake inhibitors desipramine and reboxetine, on locus coeruleus neurons from control rats and from those chronically treated with desipramine. Bath application of desipramine (1-100 microM) and reboxetine (0.1-10 microM) decreased the firing rate of locus coeruleus neurons in a concentration-dependent manner and the alpha(2)-adrenoceptor antagonist RX 821002 (10 microM) reversed these effects. In addition, reserpine (5 mg/kg, 3 h before the experiment) almost completely blocked the inhibitory effect of desipramine. Both drugs (1 microM desipramine and 0.1 microM reboxetine) potentiated the inhibitory effect of noradrenaline (10 microM). A 7-day treatment with desipramine (3 mg/kg/12 h, i.p.) caused a decrease in sensitivity to the alpha(2)-adrenoceptor agonist bromoxidine (EC(50) increased by 3.3-fold), but not to noradrenaline or reboxetine. In contrast, this treatment potentiated the inhibitory effect of desipramine with respect to control. Moreover, 14-day treatment with desipramine (3 mg/kg/12 h, i.p.) or reboxetine (10 mg/kg/12 h, i.p.) also potentiated the in vitro effect of desipramine without modifying the in vitro effect of reboxetine. These results show that desipramine and reboxetine modulate the activity of locus coeruleus neurons by noradrenaline acting on alpha(2)-adrenoceptors, and reveal that alpha(2)-adrenoceptor-independent mechanisms may also underlie the action of noradrenaline uptake inhibitors.

Animals↗

An electron microscope autoradiographic study of regenerating locus coeruleus fibers into iris tissue implants in the rat mesencephalon.

Following interruption, fibers from the locus coeruleus have been shown to undergo axonal regeneration and invade peripheral tissue implants in the rat brain. The aim of the present study was to analyze the ultrastructural features of this phenomenon using electron microscope autoradiography. For this, iris tissue implants were inserted in the region of the dorsal tegmental bundle (DTB) in the mesencephalon of unilaterally sympathectomized rats. Approximately 2 weeks later, rats were injected with [3H]-leucine in the ipsilateral locus coeruleus and sacrificed 2 days later. The tissue was prepared for EM autoradiography and studied after an incubation period of at least 12 weeks. The results showed that regenerating fibers from the locus coeruleus were most dense in regions of implant proximal to the DTB. Within the implants, less than one-half of regenerating axons present were labeled and, hence, of locus coeruleus origin. The labeled fibers were characteristically thin and unmyelinated. At this relatively short survival time, there was no evidence that the regenerating locus coeruleus axons formed synaptic specializations or contacts with the surrounding iris neuropil. Thus, while these observations confirm previous reports describing the regenerative capacity of central monoaminergic axons, they fail to provide anatomical evidence of the establishment of functional interactions between incoming fibers and the target tissue.

Afferent Pathways↗

Interactions between cholinergic and GABAergic neurotransmitters in and around the locus coeruleus for the induction and maintenance of rapid eye movement sleep in rats.

The noradrenergic "REM-off" neurons in the locus coeruleus cease firing, whereas some cholinergic and non-cholinergic "REM-on" neurons increase firing during rapid eye movement sleep. A reciprocal interaction between these neurons was proposed. However, acetylcholine did not inhibit neurons in the locus coeruleus. Nevertheless, since GABA levels increase during rapid eye movement sleep and picrotoxin injections into the locus coeruleus reduced rapid eye movement sleep, it was hypothesized that GABA in the locus coeruleus might play an intermediary inhibitory role for rapid eye movement sleep regulation. Therefore, the effects of GABA or carbachol (a mixed cholinergic agonist receptor) alone, as well as an agonist of one in presence of an antagonist of the other, in the locus coeruleus were investigated on sleep-wakefulness and rapid eye movement sleep. The cholinergic agonist carbachol increased, while the muscarinic antagonist receptor scopolamine decreased, the frequency of induction of rapid eye movement sleep per hour. In contrast, GABA and picrotoxin increased and decreased, respectively, the duration of rapid eye movement sleep per episode. However, when carbachol was injected in the presence of picrotoxin or GABA was injected in the presence of scopolamine, the effect of GABA or picrotoxin was dominant. Microinjection of both scopolamine and picrotoxin in combination reduced both the frequency of initiation as well as the duration per episode of rapid eye movement sleep. From these results we suggest that in the locus coeruleus cholinergic input modulates the frequency of induction of rapid eye movement sleep and this action is mediated through GABA interneurons, whereas the length of rapid eye movement sleep per episode is maintained by the presence of an optimum level of GABA. A model of neural connections for initiation and maintenance of rapid eye movement sleep is proposed and discussed.

Acetylcholine↗

Locus coeruleus neurons: cessation of activity during cataplexy.

Cataplexy, a symptom of narcolepsy, is a loss of muscle tone usually triggered by sudden, emotionally significant stimuli. We now report that locus coeruleus neurons cease discharge throughout cataplexy periods in canine narcoleptics. Locus coeruleus discharge rates during cataplexy were as low as or lower than those seen during rapid-eye-movement sleep. Prazosin, an alpha1 antagonist, and physostigmine, a cholinesterase inhibitor, both of which precipitate cataplexy, decreased locus coeruleus discharge rate. Our results are consistent with the hypothesis that locus coeruleus activity contributes to the maintenance of muscle tone in waking, and that reduction in locus coeruleus discharge plays a role in the loss of muscle tone in cataplexy and rapid-eye-movement sleep. Our results also show that the complete cessation of locus coeruleus activity is not sufficient to trigger rapid-eye-movement sleep in narcoleptics.

Adrenergic alpha-Antagonists↗

Internalization of mu-opioid receptors produced by etorphine in the rat locus coeruleus.

Chronic administration of mu-opioid receptor agonists is known to produce adaptive changes within noradrenergic neurons of the locus coeruleus. Although mu-opioid receptors are densely expressed by locus coeruleus neurons, the effects of acute and chronic administration of agonists on the subcellular distribution of mu-opioid receptors remain poorly understood. Therefore, we examined the ultrastructural distribution of mu-opioid receptor immunoreactivity in the locus coeruleus of rats subjected to either acute morphine, or etorphine, or chronic morphine treatment. In the locus coeruleus of control rats receiving acute saline injections or placebo pellet implants, immunogold-silver labeling for mu-opioid receptors was localized to parasynaptic and extrasynaptic portions of the plasma membranes of perikarya and dendrites. Only 8% of the gold-silver particles analyzed were distributed within the cytoplasm of dendrites and perikarya in vehicle-treated rats. Immunolabeling for mu-opioid receptors was distributed along portions of the plasma membrane that were often apposed by astroglial sheaths. After acute injections of etorphine, there was a dramatic internalization of mu-opioid receptors to intracellular compartments. Quantitative analysis of gold-silver particles indicative of mu-opioid receptors showed that a substantial number of gold particles shifted from the plasma membrane to early endosomes in dendrites from etorphine-treated rats. In dendrites sampled from etorphine-treated rats, 85% of the gold-silver grains indicative of mu-opioid receptor labeling were located in intracellular compartments as compared to 15% that were distributed along the plasma membrane. In animals that received either acute morphine injections or chronic morphine via pellet implantation, no change in the subcellular distribution of immunogold particles indicative of mu-opioid receptors was detected when compared to matched control animals. These results provide the first ultrastructural evidence that mu-opioid receptors are internalized by agonists such as etorphine, but not the partial agonist morphine, in the locus coeruleus.

Animals↗

Neuron numbers in locus coeruleus do not change with age in Fisher 344 rat.

Counts of neurons in locus coeruleus of Fisher 344 rats of ages from 12 to 32 months showed no loss with advancing age. Species comparisons of age changes in locus coeruleus neuron counts and measures of catecholamine systems suggested that the human and nonhuman primate locus coeruleus shows age changes and seen in rodent locus coeruleus. It is suggested that although rodent models of human aging have value under some circumstances, rodent and primate brain should not be considered equivalent with regard to phenomena related to noradrenergic mechanisms.

Aging↗

Abnormal postnatal ontogeny of the locus coeruleus in the epileptic mutant mouse quaking.

The tonic-clonic convulsions of the quaking mutant mice have been shown to be associated with the hyperplasia of the nucleus locus coeruleus, the origin of most brain noradrenergic neurons. In the present study, the postnatal ontogeny of the locus coeruleus has been studied by tyrosine hydroxylase immunolabeling in the mutant mice quaking and their controls at postnatal days 1, 30 and 90. In the control mice, the number of immunoreactive neuronal cell bodies increased significantly in the rostral half of the locus coeruleus between birth and postnatal day 30, while it decreased significantly in the caudal half between birth and adulthood. Thus, during postnatal maturation, the distribution of locus coeruleus neurons was shifted in the rostral direction. In the quaking mutant mice, while the increase of immunolabeling between birth and postnatal day 30 was observed in the rostral half of the locus coeruleus, no diminution could be found in the caudal half between birth and adulthood. As a result, the rostral shift of tyrosine hydroxylase immunoreactivity was not observed. Consequently, in adult mice, the caudal part of the mutants locus coeruleus appeared to contain significantly more neurons than the corresponding region in the controls. These results indicate that the hyperplasia of the locus coeruleus of the quaking mice that we had previously reported results from an alteration of the postnatal maturation of this nucleus. This developmental abnormality might be a primary determinant of the inherited epilepsy of the quaking mutant mice.

Aging↗

Effects of 6-hydroxydopamine lesions of locus coeruleus on startle in rats.

To examine the possible involvement of the norepinephrine (NE) containing neurons of the locus coeruleus in the modulation of behavioral reactivity to sensory stimulation, bilateral chemical lesions of the locus coeruleus were made by local injection of the catecholamine neurotoxin 6-hydroxydopamine. Both histochemical and biochemical analyses confirmed the effectiveness of the lesions in specifically eliminating the NE containing cell bodies of the locus coeruleus and reducing the NE content of the hippocampus and substantia nigra by 45% and 69% respectively. Rats were tested both 5 and 36 days after lesioning for their startle response to a repetitive series of tactile stimuli. On both days, locus coeruleus lesioned rats exhibited consistently reduced startle responses throughout the stimulus series. Additionally, lesioned rats showed a far more rapid rate of response habituation, particularly in the first test. The results are discussed in terms of a possible influence of the locus coeruleus on the process of sensitization to sensory stimuli.

Animals↗

Hippocampal regulation of the survival and morphological development of locus coeruleus neurons in dissociated cell culture.

The influence of target structures on neural development, originally described for the peripheral nervous system, has more recently been investigated in the central nervous system. We sought to determine whether targets regulate the development of the locus coeruleus, with its diffuse and complex projections in marked contrast to the simpler neural circuits of the peripheral nervous systems. Dissociated locus coeruleus cells were grown alone or with the hippocampus in serum-free, chemically defined medium that minimized non-neuronal growth. Coculture with the hippocampus resulted in a significant increase in locus coeruleus tyrosine hydroxylase activity. Elevated tyrosine hydroxylase activity was accompanied by a commensurate increase in the number of tyrosine hydroxylase-immunoreactive cells, suggesting hippocampal enhancement of locus coeruleus survival. Furthermore, when hippocampal cells were added to locus coeruleus dissociates at zero time, or after two days, tyrosine hydroxylase-positive cell number was increased only by hippocampal cells added initially, suggesting that the target does indeed foster survival. The apparent target enhancement of locus coeruleus survival seems to be selective since total protein and total neuron number, estimated with neuron-specific enolase immunocytochemistry, were not affected by the hippocampus. Coculture with the hippocampus also increased the length and complexity of locus coeruleus cell processes. Neither the increase in tyrosine hydroxylase cell number nor the changes in morphology could be attributed to nonspecific effects of the increased cell density in cocultures. Our observations thus suggest that the target hippocampus influences the survival and neurite elaboration of afferent locus coeruleus neurons.

Aging↗