Characterization of neurochemically specific projections from the locus coeruleus with respect to somatosensory-related barrels

Tactile information from the rodent mystacial vibrissae is relayed through the ascending trigeminal somatosensory system. At each level of this pathway, the whiskers are represented by a unique pattern of dense cell aggregates, which in layer IV of cortex are known as "barrels." Afferent inputs from the dorsal thalamus have been demonstrated repeatedly to correspond rather precisely with this modular organization. However, axonal innervation patterns from other brain regions such as the noradrenergic locus coeruleus are less clear. A previous report has suggested that norepinephrine-containing fibers are concentrated in the center/hollow of the barrel, while other studies have emphasized a more random distribution of monoaminergic projections. To address this issue more directly, individual tissue sections were histochemically processed for cytochrome oxidase in combination with dopamine-beta-hydroxylase, the synthesizing enzyme for norepinephrine, or the neuropeptide galanin. These two neuroactive agents were of particular interest because they colocalize in a majority of locus coeruleus neurons and terminals. Our data indicate that discrete concentrations or local arrays of dopamine-beta-hydroxylase- or galanin-immunoreactive fibers are not apparent within the cores of individual barrels. As such, the data suggest that cortical inputs from the locus coeruleus are not patterned according to cytoarchitectural landmarks or the neurochemical identity of coeruleocortical efferents. While transmitter-specific actions of norepinephrine and/or galanin may not be derived from the laminar/spatial connections of locus coeruleus axons, the possibility remains that the release of these substances may mediate distinctive events through the localization of different receptor subclasses, or the contact of their terminals onto cells with certain morphological characteristics or ultrastructural components.     

The mode of projections of single locus coeruleus neurons to the cerebral cortex in rats

Axonal distributions of single locus coeruleus neurons within the cerebral cortex were examined with antidromic stimulation technique combined with cortical lesions (frontal lobotomy and lobectomy). In urethan-anesthetized rats, stimulating electrodes were implanted in 10 points extending over nearly the entire cerebral cortex, and antidromic responses of single locus coeruleus neurons to stimulation of these stimulus sites were analysed. Fifty percent of locus coeruleus neurons examined were activated antidromically from at least one cortical point in the cerebral cortex. The pattern and extent of axonal distributions of single locus coeruleus neurons in the cortex appeared to vary from cell to cell. From the results obtained in rats with the cortical lesions, it is concluded that in addition to locus coeruleus neurons with intracortical axons running from rostral to caudal, there are the neurons projecting to the occipital cortex without innervating the frontal cortex and those projecting simultaneously to the frontal and occipital cortex with two axonal branches. There was no topographic order between the recording sites within the locus coeruleus and the projection sites in the cortex.     

Evidence that dorsal locus coeruleus neurons can maintain their spinal cord projection following neonatal transection of the dorsal adrenergic bundle in rats

Summary In adult rats, locus coeruleus neurons which extend axons to the spinal cord are found only at mid-rostrocaudal levels of the nucleus, where they are essentially confined to its ventral, wedge-shaped half (Satoh et al. 1980; Westlund et al. 1983; Loughlin et al. 1986). However, during early postnatal development, coeruleospinal cells are found throughout the locus coeruleus (Cabana and Martin 1984; Chen and Stanfield 1987). This developmental restriction of the distribution of coeruleospinal neurons is due to axonal elimination rather than to cell death, since neurons retrogradely labeled through their spinal axons perinatally are still present in the dorsal portion of the locus coeruleus at survival periods beyond the age at which these cells lose their spinal projection (Chen and Stanfield 1987). I now report that if axons ascending from the locus coeruleus are cut by transecting the dorsal adrenergic bundle on the day of birth, a more widespread distribution of coeruleospinal neurons is retained beyond the perinatal period. These results not only indicate that the absence of the normally maintained collateral of a locus coeruleus neuron is sufficient to prevent the elimination of a collateral which would otherwise be lost, but also may imply that during normal postnatal development the presence of the maintained collateral is somehow causally involved in the elimination of the transient collateral.     

The distribution of hippocampal and spinal projecting cells in the locus coeruleus of tottering mice

Wheat germ agglutinin conjugated to horseradish peroxidase and Fast Blue were used as retrograde tracers to examine the distribution of coeruleohippocampal and coeruleospinal somata within the locus coeruleus of normal and tottering mutant mice. The distributions of these projection neuron populations in normal mice are similar to what has been found in other species, and the distributions of these projection neurons in tottering mice are indistinguishable from those in normal mice, in spite of the norepinephrine hyperinnervation of certain locus coeruleus targets, including the hippocampus, in the tottering mutant. These observations lend support to the notion that the defect in tottering acts fairly directly on mechanisms involved in the development of locus coeruleus axonal arbors within certain target regions.     

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.     

The amygdalo-brainstem pathway: selective innervation of dopaminergic, noradrenergic and adrenergic cells in the rat

The present study investigated the organization and distribution of amygdaloid axons within the various brainstem dopaminergic, noradrenergic and adrenergic cell groups. This was accomplished via Phaseolus vulgaris leucoagglutinin lectin (PHA-L) anterograde tracing technique combined with glucose-oxidase immunocytochemistry to catecholamine markers (i.e. tyrosine hydroxylase, dopamine beta-hydroxylase, and phenylethanolamine N-methyltransferase). Injections of PHA-L within the medial part of the central amygdaloid nucleus resulted in axonal labeling within most catecholamine containing cell groups within the brainstem. The most heavily innervated catecholaminergic groups were the A9 (lateral) cells of the substantia nigra, the A8 dopaminergic cells of the retrorubral field and the C2 adrenergic cells of nucleus of the solitary tract. Amygdaloid terminals frequently contacted cells within these regions. A moderate amount of amygdaloid terminals were located within the rostral A6 (locus coeruleus) and A2 (nucleus of the solitary tract) groups. Amygdaloid terminal contacts were apparent on the majority of the rostral A6 and A2 neurons. Light or no amygdaloid terminal labeling was observed within the other brainstem catecholaminergic cell groups. Thus, the amygdala mainly innervates the A8 and lateral A9 dopaminergic cells of midbrain, rostral locus coeruleus (A6) noradrenergic neurons and the adrenergic (C2) and noradrenergic (A2) cells within the nucleus of the solitary tract. Selective innervation of these brainstem catecholaminergic systems may be important for integration of amygdaloid-mediated defensive and stress-induced behaviors.     

Inhibition of noradrenergic locus coeruleus neurons by C1 adrenergic cells in the rostral ventral medulla.

Recent anatomical and physiological experiments indicate that the nucleus locus coeruleus receives a predominant excitatory amino acid input, as well as a substantial inhibitory input, from the nucleus paragigantocellularis in the ventrolateral medulla. To determine whether C1 adrenergic neurons are involved in the inhibitory projection, the effects of the alpha-2 adrenoceptor antagonist, idazoxan, on inhibitory responses of locus coeruleus neurons to paragigantocellularis stimulation were characterized in the rat. Intravenous administration of idazoxan (0.2-1 mg/kg) attenuated paragigantocellularis-evoked inhibition, and often revealed an underlying weak excitation. Intraventricular administration of kynurenate, an excitatory amino acid antagonist, eliminated excitation from paragigantocellularis and disclosed an underlying inhibitory response in many locus coeruleus neurons that were previously excited by paragigantocellularis stimulation. These results revealed that about 90% of locus coeruleus neurons receive inhibition from the paragigantocellularis. Intravenous idazoxan significantly reduced such paragigantocellularis-evoked inhibition, completely blocking this response in three of eight locus coeruleus cells tested. Idazoxan was much more potent when locally infused into the locus coeruleus. Local infusion of idazoxan (0.1-2.5 ng) into locus coeruleus produced a dose-dependent decrease of paragigantocellularis-evoked inhibition and completely blocked the inhibition in 10/33 locus coeruleus neurons, indicating that the site of idazoxan action was in the locus coeruleus. These results extend our previous anatomical studies of adrenergic input to locus coeruleus, and indicate that C1 adrenergic neurons in the paragigantocellularis provide a direct inhibitory input to the great majority of locus coeruleus noradrenergic neurons. In addition, this is the first report of a neuronal response to activation of C1 adrenergic cells indicating that these neurons are strongly inhibitory when acting at alpha-2 receptors in vivo.     

Locus coeruleus projections to the dorsal motor vagus nucleus in the rat.

The origin of the noradrenergic innervation of the preganglionic autonomic nuclei in the medulla oblongata and spinal cord is still controversial. In this investigation descending connections of the locus coeruleus to the dorsal motor vagus nucleus in the rat are studied with Phaseolus vulgaris leucoagglutinin and horseradish peroxidase as neuroanatomical tracers. Locus coeruleus projections in the motor vagus nucleus are found in the medial part at rostral levels and in the lateral part at intermediate levels of this nucleus. The terminal labeling in the lateral intermediate part of the vagus nucleus appears in an area where possibly preganglionic parasympathetic cardiac neurons are located, suggesting that the locus coeruleus might be involved in regulation of cardiovascular functions. After small iontophoretic injections of horseradish peroxidase in the motor vagus nucleus, retrogradely labeled cells are found in the ventral part of the locus coeruleus and occasionally in the dorsal part of the nucleus. The results show that the locus coeruleus-dorsal motor vagus nucleus pathway may participate in the inhibition of the cardiac preganglionic neurons in the dorsal motor vagus nucleus by the hypothalamic paraventricular nucleus.     

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus

Locus coeruleus neurons are strongly coupled during early postnatal development, and it has been proposed that these neurons are linked by extraordinarily abundant gap junctions consisting of connexin32 (Cx32) and connexin26 (Cx26), and that those same connexins abundantly link neurons to astrocytes. Based on the controversial nature of those claims, immunofluorescence imaging and freeze-fracture replica immunogold labeling were used to re-investigate the abundance and connexin composition of neuronal and glial gap junctions in developing and adult rat and mouse locus coeruleus. In early postnatal development, connexin36 (Cx36) and connexin43 (Cx43) immunofluorescent puncta were densely distributed in the locus coeruleus, whereas Cx32 and Cx26 were not detected. By freeze-fracture replica immunogold labeling, Cx36 was found in ultrastructurally-defined neuronal gap junctions, whereas Cx32 and Cx26 were not detected in neurons and only rarely detected in glia. In 28-day postnatal (adult) rat locus coeruleus, immunofluorescence labeling for Cx26 was always co-localized with the glial gap junction marker Cx43; Cx32 was associated with the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase); and Cx36 was never co-localized with Cx26, Cx32 or Cx43. Ultrastructurally, Cx36 was localized to gap junctions between neurons, whereas Cx32 was detected only in oligodendrocyte gap junctions; and Cx26 was found only rarely in astrocyte junctions but abundantly in pia mater. Thus, in developing and adult locus coeruleus, neuronal gap junctions contain Cx36 but do not contain detectable Cx32 or Cx26, suggesting that the locus coeruleus has the same cell-type specificity of connexin expression as observed ultrastructurally in other regions of the CNS. Moreover, in both developing and adult locus coeruleus, no evidence was found for gap junctions or connexins linking neurons with astrocytes or oligodendrocytes, indicating that neurons in this nucleus are not linked to the pan-glial syncytium by Cx32- or Cx26-containing gap junctions or by abundant free connexons composed of those connexins.     

Prostaglandin EP3 receptor protein in serotonin and catecholamine cell groups: a double immunofluorescence study in the rat brain

Prostaglandin E(2) exerts diverse physiological actions in the central nervous system with unknown mechanisms. We have reported the immunohistochemical localization of the EP3 receptor, one of the prostaglandin E receptor subtypes, in various brain regions including many monoaminergic nuclei. In the present study, a double immunofluorescence technique with an antibody to EP3 receptor and antibodies to markers for monoamine neurons was employed to examine the expression of the receptor in serotonin and catecholamine neurons, and to reveal the distribution of the receptor-expressing monoamine neurons in the rat brain. Almost all serotonergic cells in the medulla oblongata (B1-B4) exhibited EP3 receptor-like immunoreactivity, whereas mesencephalic and pontine serotonergic cell groups (B5-B9) contained relatively small populations of EP3 receptor-immunoreactive cells. In the catecholaminergic cell groups, many of the noradrenergic A7 cells in the subcoeruleus nucleus showed immunoreactivity for the receptor. The locus coeruleus exhibited EP3 receptor-like immunoreactivity densely in the neuropil and occasionally in neuronal cell bodies, all of which were immunopositive for dopamine beta-hydroxylase, as observed by confocal laser microscopy. Many of the other noradrenergic and adrenergic cell groups contained small populations of EP3 receptor-like immunoreactive cells. In contrast, no EP3 receptor-like immunoreactivity was detected in the noradrenergic A2 and A4, the adrenergic C2, and all the dopaminergic cell groups.The expression of EP3 receptor by most of the serotonergic, noradrenergic and adrenergic cell groups suggests that prostaglandin E(2) modulates many physiological processes mediated by widely distributed monoaminergic projections through activation of the EP3 receptor on the monoaminergic neurons; for instance, it may modulate nociceptive and autonomic processes by affecting the descending serotonergic pathway from the raphe magnus nucleus to the spinal cord.     

The cerebellar projection from locus coeruleus as studied with retrograde transport of horseradish peroxidase in the cat.

The cerebellar afferent projection from locus coeruleus has been studied in the cat by means of retrograde axonal transport of horseradish peroxidase. Labelled cells are present bilaterally in locus coeruleus only following injections in the cerebellar vermis (especially its anterior and posterior parts), the ventral paraflocculus and the flocculus. The labelled cells are restricted to the caudal half of the nucleus. A few labelled cells are also present in locus coeruleus following injections in the fastigial nucleus, and in nucleus interpositus anterior. The findings are discussed in relation to other studies on the efferent and afferent connections of the locus coeruleus.     

Synchronous bursting of locus coeruleus neurons in tissue culture

Synchronous bursts of firing of locus coeruleus neurons have been observed in unanesthetized rats, particularly in response to various sensory stimuli. In explant tissue cultures, synchronous bursting activity of locus coeruleus neurons was also observed and the possible mechanisms responsible for this synchronous activation have been investigated. Barrages of depolarizing events apparently initiated and continued throughout spontaneous bursts of spikes in the cultured neurons. Simultaneous intracellular recordings from pairs of neurons show a very high degree of synchrony of such barrages between cells. On the basis of tests for electrical coupling in simultaneously recorded cell pairs, and tests for dye coupling with Lucifer Yellow, it was concluded that the synchrony is not due to electrical coupling of locus coeruleus neurons. Small non-synaptic interactions between cell pairs that may reflect elevated extracellular potassium levels have been observed on some occasions. Spontaneous and evoked depolarizations similar to those initiating the bursts appear to be synaptically mediated events, suggesting that locus coeruleus neurons are synchronously activated by a common excitatory input. It was concluded that the neurons providing this common excitation are located within or very close to the locus coeruleus, at least at birth. The synchronization of activation of many locus coeruleus neurons could result in almost simultaneous release of neurotransmitter in the widespread target areas of locus coeruleus projections.     

Evidence that 3,3',5-triiodothyronine is concentrated in and delivered from the locus coeruleus to its noradrenergic targets via anterograde axonal transport.

Recent immunohistochemical studies of rat brain triiodothyronine reveal heaviest localization in locus coeruleus perikarya. The cellular distribution is similar to that observed in concomitant studies of tyrosine hydroxylase immunohistochemistry: heavy clumps of immunoreactive triiodothyronine are distributed within locus coeruleus cytosol and in cell processes, leaving cell nuclei unstained. At the same time, in locus coeruleus targets, cell nuclei as well as surrounding neuropil are prominently triiodothyronine labeled. These observations, combined with diverse evidence linking thyroid hormone with norepinephrine at many levels of physiological and pathophysiological function, led to the hypothesis that the locus coeruleus binds and accumulates triiodothyronine and delivers the hormone via anterograde axonal transport to postsynaptic locus coeruleus targets, where nuclear triiodothyronine receptors are densely concentrated. Furthermore, the hypothesis predicts that destruction of locus coeruleus nerve terminals would interrupt this neural route of triiodothyronine delivery and prevent or reduce triiodothyronine labeling of nuclear receptors in noradrenergic target cells. To test this formulation, we gave the specific locus coeruleus lesioning agent, N-(2-chloroethyl)-N-2-bromobenzylamine hydrochloride (DSP-4), to adult male rats and examined their brains three, five and seven days thereafter by triiodothyronine and, in alternate sections, tyrosine hydroxylase immunohistochemistry. Treatment with DSP-4 resulted in specific and selective reduction in tyrosine hydroxylase and triiodothyronine immunohistochemical labeling in cell nuclei and in nerve cell processes within the neuropil of the hippocampus and cerebral cortex at all time periods examined. The results demonstrate that full occupancy of locus coeruleus target cells by triiodothyronine requires the presence of intact locus coeruleus projections and supports the proposal that, like norepinephrine, triiodothyronine delivery to noradrenergic targets occurs through delivery by locus coeruleus terminals. These findings provide strong support for earlier proposals that triiodothyronine functions as a co-transmitter with norepinephrine in addition to or as part of its genomic role in the cells receiving noradrenergic innervation.     

Do noradrenergic descending tract fibres contribute to the depression of transmission from group II muscle afferents following brainstem stimulation in the cat?

Two alpha 2 noradrenaline antagonists, idazoxan and yohimbine, were injected in midlumbar segments of the spinal cord to test whether they counteract depression of field potentials evoked by group II muscle afferents by conditioning stimuli applied in the brainstem. The tested field potentials were those evoked monosynaptically in the intermediate zone of midlumbar segments. Their depression reflected thus the depression of transmission between group II fibres and their first relay neurones. The conditioning stimuli were applied either within the ipsilateral locus coeruleus/subcoeruleus or outside these nuclei (in the raphe magnus, raphe obscurus, or cuneiform nuclei). The brainstem evoked depression of the tested field potentials (n = 12) was reduced following injection of idazoxan or yohimbine to about two thirds of that which was evoked originally but in three cases to about one half. The study leads thus to the conclusion that noradrenergic descending tract neurones contribute to the depression of transmission from group II afferents to spinal interneurones and that such noradrenergic neurones are activated by stimuli applied within as well as outside their nuclei. However, the relative contribution of monoaminergic and non-monoaminergic descending tract neurones to the control of transmission from group II afferents to these neurones remains to be established.     

Non-dopaminergic catecholaminergic neurons of mesencephalic and medullary nuclei contain different levels of dopamine

The present study addresses the question whether metabolic dopamine can be immunocytochemically detected within nondopaminergic catecholaminergic axonal fibers. For this purpose, confocal microscopy was used to analyze sections treated for the double fluorescence immunostaining of dopamine and either noradrenaline or phenylethanolamine-N- methyltransferase (the enzyme in adrenergic neurons that converts noradrenaline into adrenaline). Our data demonstrate that throughout the brain and spinal cord, the majority of the axonal fibers that reacted with the anti-phenylethanolamine-N-methyltansferase antibodies also exhibited faint to intense dopamine immunoreactivity. Similarly noradrenaline and dopamine immunoreactivities were frequently colocalized within axonal fibers innervating brain and spinal cord regions that receive a dense innervation from medullary noradrenergic neurons. On the contrary, dopamine was rarely detected within noradrenaline-immunoreactive fibers in those regions where the nomdrenergic innervation essentially arises from noradrenergic neurons of the locus coeruleus. A similar differential dopamine immunostaining was observed in the corresponding neuronal perikarya of the medulla oblongata and the locus coeruleus. These data indicate that two types of non-dopaminergic catecholaminergic neurons can be distinguished according to their content in dopamine: (i) the noradrenergic and adrenergic neurons located in the medulla oblongata, whose cell bodies and axons contain high concentrations of metabolic dopamine and (ii) the noradrenergic neurons located in the mesencephalon, which contain low levels of metabolic dopamine.     

Influence of reserpine administration on neuropeptide Y immunoreactivity in the locus coeruleus and caudate-putamen nucleus of the rat brain.

The effects of treatment with reserpine (10 mg/kg, i.p.) a monoamine depleting agent, on neuropeptide Y immunoreactivity were studied immunohistochemically in neurons of two rat brain structures: locus coeruleus and caudate-putamen nucleus. It was found that reserpine after 24 h increased neuropeptide Y immunoreactivity level but no significant changes were observed 4 and 72 h or 5 days after the injection. The results indicate that despite the known co-existence of neuropeptide Y and noradrenaline in some neurons of the locus coeruleus no concomitant decrease in neuropeptide Y immunoreactivity level was found after reserpine when noradrenaline was depleted from nerve cell bodies and terminals. The increase in neuropeptide Y immunoreactivity observed 24 h after reserpine injection may suggest that the neuropeptide Y-containing neuronal systems of the locus coeruleus and caudate-putamen nucleus are controlled by monoaminergic afferents.     

Prazosin,an α1-adrenoceptor antagonist,prevents memory deterioration in the APP23 transgenic mouse model of Alzheimer's disease

Noradrenergic deficits have been described in the hippocampus and the frontal cortex of Alzheimer's disease brains, which are secondary to locus coeruleus degeneration. Locus coeruleus is the brain stem nucleus responsible for synthesis of noradrenaline and from where all noradrenergic neurons project. In addition, it has been suggested that noradrenaline might play a role in modulating inflammatory responses in Alzheimer's disease. In this study we aimed to investigate the effect of various agonists and antagonists for adrenergic receptors on amyloid precursor protein processing. Among them, we found that prazosin, an α₁-adrenoceptor antagonist, was able to reduce the generation of amyloid β in N2a cells. Treatment of transgenic APP23 mice with prazosin prevented memory deficits over time. Although prazosin did not influence amyloid plaque load, it induced astrocytic proliferation and increased the release of apolipoprotein E and anti-inflammatory cytokines. These findings suggest that chronic treatment with prazosin leads to an anti-inflammatory response with potential beneficial effects on cognitive performance.     

Age-dependent changes in axonal branching of single locus coeruleus neurons projecting to two different terminal fields

Age-dependent changes in the axonal branching patterns of single locus coeruleus neurons, which innervate both the frontal cortex and hippocampus dentate gyrus, have been studied in male F344 rats. We used an electrophysiological approach involving antidromic activation to differentiate single from multi-threshold locus coeruleus neurons in each terminal field with age (7-27 mo of age). Most of these neurons have a single threshold in the young rats, whereas in the older brains, the neurons have multi-threshold responses. This implies an increased amount of axonal branching in the older brains. The time course of the increase differs in the two terminal fields, suggesting that the degree of plasticity or age-dependent increase in branching can differ across terminal fields.     

Fluorescence histochemical and neurohistological investigations on the locus coeruleus of the rat (author's transl)

The Locus coeruleus (LC) of the adult rat was investigated by means of fluorescence histochemical and rapid GOLGI impregantion technique. The majority of LC neurons displayed, as a result of application of the FALCK-HILLARP-technique and the LOREN-technique, resp, the well-known fluorescence typical of catecholamine neurons. There was lack of fluorescence within a minor portion of cells. Fluorescence histochemically, serotoninergic afferents could be shown to go up to noradrenaline-containing neurons in some cases. In the GOLGI material, 3 types of neurons could be distinguished: Polygonal neurons exhibiting somatic spines, fusiform neurons, and small-sized spine-less neurons. Based on morphological features, the polygonal neuron type is considered to represent the monoamine-containing neurons of the Locus coeruleus. It appears uncertain at present to attribute the fusiform and small spineless neuron types in a functional manner. functional implications inherent that morphological heterogeneity have been discussed.