Opposing effects of hypoxia on catecholaminergic locus coeruleus and hypocretin/orexin neurons in chick embryos

Terrestrial vertebrate embryos face a risk of low oxygen availability (hypoxia) that is especially great during their transition to air‐breathing. To better understand how fetal brains respond to hypoxia, we examined the effects of low oxygen availability on brain activity in late‐stage chick embryos (day 18 out of a 21‐day incubation period). Using cFos protein expression as a marker for neuronal activity, we focused on two specific, immunohistochemically identified cell groups known to play an important role in regulating adult brain states (sleep and waking): the noradrenergic neurons of the Locus Coeruleus (NA‐LC), and the Hypocretin/Orexin (H/O) neurons of the hypothalamus. cFos expression was also examined in the Pallium (the avian analog of the cerebral cortex). In adult mammalian brains, cFos expression changes in a coordinated way in these areas. In chick embryos, oxygen deprivation simultaneously activated NA‐LC while deactivating H/O‐producing neurons; it also increased cFos expression in the Pallium. Activity in one pallial primary sensory area was significantly related to NA‐LC activity. These data reveal that at least some of the same neural systems involved in brain‐state control in adults may play a central role in orchestrating prenatal hypoxic responses, and that these circuits may show different patterns of coordination than seen in adults. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 74: 1030–1037, 2014     

Development of catecholaminergic phenotypic characters in the mouse locus coeruleus in vivo and in culture

While abundant studies have begun to elucidate ontogeny of the peripheral nervous system, molecular mechanisms underlying brain development remain obscure. To approach this problem, we initiated parallel in vivo and in vitro studies of the mouse locus coeruleus (l.c.), a brainstem noradrenergic nucleus. The catecholaminergic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) were used to monitor phenotype expression and development. TH catalytic activity and immunocytochemical reactivity were initially detectable on gestational Day 13 (E-13) in vivo, and adult levels of activity were approximately by the third postnatal week. Immunotitration studies indicated that the developmental increase was due to accumulation of enzyme molecules and not enzyme activation. The in vivo developmental profile of DBH approximated that of TH. To begin defining regulatory mechanisms, explants of embryonic brainstem were placed in culture. Explantation on E-12, prior to expression of TH or DBH, resulted in the de novo appearance of these phenotypic characters after 4 days. Explantation on E-18, after the enzymes are already expressed, was followed by a striking sixfold rise in TH activity. Immunotitration studies revealed that the increase in TH activity in E-18 cultures was attributable to increased molecule number, reproducing the in vivo results. Moreover, the E-18 explants, cultured for 3 weeks, attained higher plateau levels of TH activity than E-12 cultures, and this differences was due to increased molecule number. Morphometric analysis indicated that 3-week E-12 cultures actually had more l.c. cells than E-18 cultures, indicating that differences in TH were not due to increased cells in the E-18 l.c. Finally, systemic study revealed that the development of TH activity in culture increased progressively from E-11 to E-12 to E-13, suggesting that critical regulatory events occur at this time. Our studies suggest that the l.c. is an excellent model for the study of brain development in vivo and in vitro. Initial phenotypic expression and dramatic development occur in culture in the absence of normal targets, normal afferent innervation and, presumably, normal humoural milieu.     

Topography of catecholamine-containing neurons of the locus coeruleus in the cat

Topography of catecholamine-containing (CA) neurons of the cat locus coeruleus was studied using a combination of the catecholamine histofluorescence method and rapid embedding of the brain tissue into the paraffin wax. The distribution of CA neurons was examined at frontal and sagittal sections of the brain stem. Unlike that shown previously the quantity of CA neurons in the rostral pole of the locus coeruleus was somewhat higher while at the frontal level of P--2.0-P--4.0 the significant number of CA cells of the locus coeruleus was localized more ventromedially.     

Ultrastructure of the parietal region of the brain after selective damage of the serotoninergic system

The selective lesion of the afferent serotoninergic system by means of intracisternal administration of 5,7-dihydroxytryptamine produces certain structural rearrangements in a part of cellular elements and in various parts of the neuropil of the neocortical parietal area. The deficiency of serotoninergic innervation influences the metabolic processes. This is evident from the change in the structure of protein synthesizing apparatus in a number of postsynaptic neurons. It is possible that the structural rearrangements observed in the serotoninergic and in the conjugated to it mediatory systems make the base of functional disorders in the neocortex and are accompanied with certain changes in the integrative activity of the brain.     

Morphological restructuring of the cellular elements of the ventral tegmental area in selective damage to the catecholaminergic systems of the brain

Electron microscopical investigation of the ventral tegmental area at various time after administration of a specific neurotoxin 6-hydroxydopamine made it possible not only to reveal catecholamine reactive elements, but also those structural reorganizations in them, that are dependent on the lesion of the catecholaminergic systems, including certain changes in the receptor apparatus. Dark neurons with the change of different organelles preserve for a long time, up to 2.5 months. Reactive and destructive reorganizations of some axons, terminal buttons and postsynaptic poles of the ventral tegmental area are accompanied with certain functional disturbances, observed both experimentally and at a pathological process. The results of the work demonstrate that under conditions of the model experiments there is a possibility to study various stages of pathogenesis of a number of nervous-psychical diseases, connected or accompanied with disorders in certain mediator systems.     

Spontaneous unit activity of locus coeruleus neurons after destruction of some nuclei of the medulla oblongata

Bilateral lesions of the nuclei prepositus hypoglossi produced a more than twofold decrease in the mean frequency discharges in the neurons of the nucleus coeruleus. The number of neurons with burst activity and the number of polymodal neurons substantially increased. Lesion of the nucleus tractus solitarius resulted in an increase in the number of neurons with regular activity and certain decrease in the mean discharge frequency of coeruleus neurons. The results confirm the suggestion about a substantial role of the nucleus prepositus hypoglossi in relaying afferent effects to the activity of locus coeruleus neurons.     

BMP-2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin-3 (NT-3) and also glial cell line- derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum-free conditions, none of the factors tested, including NT-3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein-2 (BMP-2), promoted the survival of tyrosine hydroxylase (TH)-immunoreactive neurons at 6 days in vitro. However, when BMP-2 was coadministered with any of these factors the number of LC TH-positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT-3, bFGF, or BMP-2. The strongest effect (a fourfold increase in the number of TH-positive cells) was induced by cotreatment with forskolin, BMP-2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors.     

Potentiation of the glutamatergic synaptic input to rat locus coeruleus neurons by P2X7 receptors

Locus coeruleus (LC) neurons in a rat brain slice preparation were superfused with a Mg²⁺-free and bicuculline-containing external medium. Under these conditions, glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) were recorded by means of the whole-cell patch-clamp method. ATP, as well as its structural analogue 2-methylthio ATP (2-MeSATP), both caused transient inward currents, which were outlasted by an increase in the frequency but not the amplitude of the sEPSCs. PPADS, but not suramin or reactive blue 2 counteracted both effects of 2-MeSATP. By contrast, α,β-methylene ATP (α,β-meATP), UTP and BzATP did not cause an inward current response. Of these latter agonists, only BzATP slightly facilitated the sEPSC amplitude and strongly potentiated its frequency. PPADS and Brilliant Blue G, as well as fluorocitric acid and aminoadipic acid prevented the activity of BzATP. Furthermore, BzATP caused a similar facilitation of the miniature (m)EPSC (recorded in the presence of tetrodotoxin) and sEPSC frequencies (recorded in its absence). Eventually, capsaicin augmented the frequency of the sEPSCs in a capsazepine-, but not PPADS-antagonizable, manner. In conclusion, the stimulation of astrocytic P2X7 receptors appears to lead to the outflow of a signalling molecule, which presynaptically increases the spontaneous release of glutamate onto LC neurons from their afferent fibre tracts. It is suggested, that the two algogenic compounds ATP and capsaicin utilise separate receptor systems to potentiate the release of glutamate and in consequence to increase the excitability of LC neurons.     

Effects of cocaine on the electrical activity of single noradrenergic neurons from locus coeruleus

The effects of intravenous (i.v.) cocaine HCl on single identified spontaneously firing noradrenergic neurons in the nucleus locus coeruleus (LC) were studied in rats in vivo. Cocaine (0.25-1 mg/kg) produced inhibition of spontaneously firing LC neurons, which was reversed by the administration of the selective alpha 2-adrenoceptor antagonist, piperoxane (250 micrograms/kg, i.v.). Procaine, a local anesthetic that is structurally related to cocaine, did not inhibit LC neurons in doses up to 4 mg/kg, i.v. These results suggest that cocaine in low doses has significant central sympathomimetic effects at the single noradrenergic neuron level and that the inhibition of spontaneous activity may be mediated by alpha 2-adrenoceptors. Our results also indicate that cocaine in pharmacologically relevant doses, can significantly affect central alpha 2-adrenoceptor regulatory processes.     

Arecoline excites rat locus coeruleus neurons by activating the M2-muscarinic receptor

The action of arecoline on rat locus coeruleus neurons was studied by intracellular recording from the in vitro brain slice preparation. Superfusion of arecoline (0.1-100 microM) caused two dose-related effects, an increased firing rate and, in neurons previously hyperpolarized to a constant potential by passing a steady hyperpolarizing current across the membrane, depolarization. Both effects were associated with a reduction in membrane input resistance. Moreover, the arecoline-induced excitatory effects were antagonized by the muscarinic receptor antagonist, atropine, but not by the nicotinic receptor antagonist, hexamethonium. Methoctramine, a selective M2-muscarinic receptor antagonist, was also effective in reversing the arecoline-induced effects, with a dissociation equilibrium constant of 14.2+/-1.2 nM (n=6). These results therefore suggest that arecoline exerts its excitatory actions by binding to M2-muscarinic receptors on the cell membrane of neurons of the locus coeruleus.     

Somatic vs. dendritic responses to hypercapnia in chemosensitive locus coeruleus neurons from neonatal rats

Cardiorespiratory control is mediated in part by central chemosensitive neurons that respond to increased CO₂ (hypercapnia). Activation of these neurons is thought to involve hypercapnia-induced decreases in intracellular pH (pH_i). All previous measurements of hypercapnia-induced pH_i changes in chemosensitive neurons have been obtained from the soma, but chemosensitive signaling could be initiated in the dendrites of these neurons. In this study, membrane potential (V_m) and pH_i were measured simultaneously in chemosensitive locus coeruleus (LC) neurons from neonatal rat brain stem slices using whole cell pipettes and the pH-sensitive fluorescent dye pyranine. We measured pH_i from the soma as well as from primary dendrites to a distance 160 µm from the edge of the soma. Hypercapnia [15% CO₂, external pH (pH_o) 7.00; control, 5% CO₂, pH_o 7.45] resulted in an acidification of similar magnitude in dendrites and soma (0.26 pH unit), but acidification was faster in the more distal regions of the dendrites. Neither the dendrites nor the soma exhibited pH_i recovery during hypercapnia-induced acidification; but both regions contained pH-regulating transporters, because they exhibited pH_i recovery from an NH₄Cl prepulse-induced acidification (at constant pH_o 7.45). Exposure of a portion of the dendrites to hypercapnic solution did not increase the firing rate, but exposing the soma to hypercapnic solution resulted in a near-maximal increase in firing rate. These data show that while the pH_i response to hypercapnia is similar in the dendrites and soma, somatic exposure to hypercapnia plays a major role in the activation of chemosensitive LC neurons from neonatal rats. acid; brain stem; intracellular pH; pyranine; respiratory control; whole cell     

Distribution of norepinephrine-containing neurons projecting to the parietal association cortex and spinal cord in the cat locus coeruleus

Distribution of neurons forming projections to the parietal association cortex and spinal cord in the cat locus coeruleus (LC) was investigated by means of horseradish peroxidase retrograde transport and catecholamine histofluorescence techniques. Neurons projecting to the parietal cortex were found to be located mainly dorsally within the LC; largest numbers were observed on frontal plane P-1.0. Cells forming projections to the spinal cord were found in the ventral locus coeruleus; highest numbers of these were noted on frontal plane P-3.0. Labeled neurons were also identified in the midbrain reticular formation, pons, and medulla when applying horseradish peroxidase to the parietal cortex and spinal cord. Neurons projecting to the neocortex and spinal cord make up two different populations in the locus coeruleus, indistinguishable on grounds of neuronal morphological characteristics. It was concluded that the cat parietal association cerebral cortex, in common with the spinal cord, receives direct afferent inputs from the locus coeruleus and the reticular formation.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 112–121, January–February, 1989.     

Retrograde axonal transport specificity in the locus coeruleus neurons after [H]noradrenaline injection into the rat olfactory bulb

Retrograde axonal transport process was investigated in the afferent systems to the rat olfactory bulb, after [³H]noradrenaline ([³H]NA) injection into the olfactory bulb, in order to provide evidence regarding its specificity and the biochemical basis supporting this specificity.

Radioautographs showed that [³H]NA unilaterally injected into the olfactory bulb at a concentration of 10⁻³ M, resulted in labeling of the structures afferent to the olfactory bulb, mainly on the injected side: locus coeruleus (LC), dorsal and central raphes, nucleus of the lateral olfactory tract and piriform cortex. Heavy labeling was observed on the noradrenergic LC cell bodies, whereas the radioautographic reaction was less intense on the other structures. After 10⁻⁴ M injection, the labeling intensity of the LC cell bodies was lower while very rare weakly labeled cell bodies persisted in the dorsal raphe, nucleus of the lateral olfactory tract and piriform cortex. The LC cell bodies were exclusively labeled when the concentration of [³H]NA injection was 10⁻⁵ M. All the other structures were devoid of labeling. It was still possible to detect labeled cell bodies in the LC for a 10⁻⁶ M concentration.

Following bilateral injections of [³H]NA (10⁻³ M) the total radioactivity retrogradely transported to the LC represented about 4 times the total radioactivity measured in the periaqueductal gray substance (as control tissue of the tracer diffusion). Fractional study by ethanol of LC tissue homogenate and liquid scintillation counting of each fraction showed that 60% of the total radioactivity (about 2.5 times the control value) was in the supernatant and 40% (about 20 times the control value) was associated with the precipitate. In the other regions such as the dorsal and central raphes and periaqueductal gray substance, radioactivity was mainly found in the supernatant, except for the dorsal raphe whose precipitate contained a low amount of radioactivity (about 4 times the control value).

Colchicine (an axonal transport inhibitor) bilaterally injected into the medial forebrain bundle and systemic administration of desipramine (a noradrenaline uptake inhibitor) decreased the radioactivity associated with the LC precipitate by 90 and 85% and the LC supernatant radioactivity by 55 and 35%, respectively. These pretreatments did not significantly affect the radioactivity amounts measured in the different fractions of dorsal and central raphes and periaqueductal gray substance. Radioautographic study after colchicine treatment showed a large decrease in the labeling intensity of the LC cell bodies as compared to the non-treated side.

Therefore, we suggest that low concentrations (10⁻⁵ M) of [³H]NA injected in the olfactory bulb determine specific conditions of noradrenergic pathway labeling. This specific labeling after migration could be supported by the radioactive ethanol precipitate which would appear to contain [³H]NA- and/or ³H-derivatives-binding protein. Such a ³H-macromolecular complex, which could represent the specific carrier, may well undergo retrograde transport from the nerve terminals towards the cell bodies.     

Afferent pathway from locus coeruleus to the nucleus solitarius

After the destruction of the nucleus tractas solitarii, just caudally to the writing pen by means of a stereotaxic instrument, the system of afferent fibres to the nucleus in question was investigated by the methods of Nauta and Fink--Heimer. The fibre terminals were revealed near locus coeruleus. Investigation of the locus coeruleus by Golgi method demonstrated that it usually has neurons of reticular type and transitional ones which resemble by their form the neurons specific for sensory formations. It is possible to conclude that locus coeruleus posesses connections of visceral origin which may play a part in the afferent influence of locus coeruleus on the brain cortex.     

Characterization of a developmentally transient adrenergic depolarizing response in cultured locus coeruleus neurons

The effects of iontophoretically applied noradrenaline have been tested on intracellularly recorded locus coeruleus neurons grown in explant cultures from neonatal mice. In addition to hyperpolarizing responses mediated by alpha 2-adrenergic receptors, as observed in locus coeruleus neurons in vivo and in brain slices from adult animals, alpha 1-mediated depolarizations were observed to succeed the initial hyperpolarizations in some cultures. It was shown that the depolarizing responses were only present in younger cultures, i.e., less than 26 days in vitro. In cultures less than 20 days old, all cells displayed the biphasic hyperpolarizing-depolarizing responses. Both components of the response appear to be direct, since they were present when synaptic transmission was blocked by including tetrodotoxin or by altering divalent cations in the perfusate. The depolarizing responses were frequently reduced in solutions with altered divalent cation content, and this might reflect a calcium dependency of this response. The hyperpolarizing and depolarizing components of the responses to noradrenaline were progressively blocked by increasing concentrations of the selective antagonists yohimbine and prazosin, respectively, in the dose ranges of 100 mM - 1 microM (yohimbine) and 20-200 nM (prazosin). Recent results from electrophysiological studies of locus coeruleus neurons in brain slices suggest that similar changes occur in the animal as well as in culture. It is possible that the transient depolarizing responses reflect a developmentally important enhanced responsiveness of locus coeruleus neurons during the early postnatal period.     

A single dose of cocaine potentiates glutamatergic synaptic transmission onto locus coeruleus neurons

The brainstem locus coeruleus (LC), the primary norepinephrinergic (NE) nucleus in the brain, has been implicated in the abuse of drugs such as opioids. However, whether and how the LC-NE system is involved in cocaine addiction remains elusive. Here, we demonstrated cocaine-evoked synaptic plasticity of glutamatergic transmission onto LC neurons as one of the earliest traces occurring after a single injection of cocaine. Twenty-four hours after mice were injected intraperitoneally with cocaine, the evoked α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) mediated synaptic transmission onto LC neurons were strongly potentiated without major effect on N-methyl-d-aspartate receptor (NMDAR) mediated synaptic transmission. Compared with saline-pretreated mice, AMPAR-mediated excitatory postsynaptic currents (EPSCs) of cocaine-pretreated mice showed a marked inward rectification, demonstrating the insertion of GluR2-lacking AMPARs to plasma membrane. In addition, the single injection of cocaine did not affect presynaptic glutamate release probability measured by paired pulse ratio. Furthermore, we found that the cocaine-induced potentiation of AMPAR EPSCs could be blocked by prazosin, an inhibitor of α1-adrenoreceptor (AR), indicating that cocaine increases AMPAR transmission via α1-ARs. These results reveal that LC-NE serves as an initial target of drug intake.     

BMP‐2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin‐3 (NT‐3) and also glial cell line‐ derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum‐free conditions, none of the factors tested, including NT‐3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein‐2 (BMP‐2), promoted the survival of tyrosine hydroxylase (TH)‐immunoreactive neurons at 6 days in vitro. However, when BMP‐2 was coadministered with any of these factors the number of LC TH‐positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT‐3, bFGF, or BMP‐2. The strongest effect (a fourfold increase in the number of TH‐positive cells) was induced by cotreatment with forskolin, BMP‐2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 291–304, 2002; DOI 10.1002/neu.10034