Cultured Locus Coeruleus Neurons Research Articles

Interaction Sites of the G Protein β Subunit with Brain G Protein-coupled Inward Rectifier K+ Channel

G protein-coupled inward rectifier K(+) channels (GIRK channels) are activated directly by the G protein betagamma subunit. The crystal structure of the G protein betagamma subunits reveals that the beta subunit consists of an N-terminal alpha helix followed by a symmetrical seven-bladed propeller structure. Each blade is made up of four antiparallel beta strands. The top surface of the propeller structure interacts with the Galpha subunit. The outer surface of the betagamma torus is largely made from outer beta strands of the propeller. We analyzed the interaction between the beta subunit and brain GIRK channels by mutating the outer surface of the betagamma torus. Mutants of the outer surface of the beta(1) subunit were generated by replacing the sequences at the outer beta strands of each blade with corresponding sequences of the yeast beta subunit, STE4. The mutant beta(1)gamma(2) subunits were expressed in and purified from Sf9 cells. They were applied to inside-out patches of cultured locus coeruleus neurons. The wild type beta(1)gamma(2) induced robust GIRK channel activity with an EC(50) of about 4 nm. Among the eight outer surface mutants tested, blade 1 and blade 2 mutants (D1 and CD2) were far less active than the wild type in stimulating GIRK channels. However, the ability of D1 and CD2 to regulate type I and type II adenylyl cyclases was not very different from that of the wild type beta(1)gamma(2). As to the activities to stimulate phospholipase Cbeta(2), D1 was more potent and CD2 was less potent than the wild type beta(1)gamma(2). Additionally we tested four beta(1) mutants in which mutated residues are located in the top Galpha/beta interacting surface. Among them, mutant W332A showed far less ability than the wild type to activate GIRK channels. These results suggest that the outer surface of blade 1 and blade 2 of the beta subunit might specifically interact with GIRK and that the beta subunit interacts with GIRK both over the outer surface and over the top Galpha interacting surface.

Opposing mechanisms of regulation of a G-protein-coupled inward rectifier K+ channel in rat brain neurons.

In locus coeruleus neurons, substance P (SP) suppresses an inwardly rectifying K+ current via a pertussis toxin-insensitive guanine nucleotide binding protein (G protein; GnonPTX), whereas somatostatin (SOM) or [Met]enkephalin (MENK) enhances it via a pertussis toxin-sensitive G protein (GPTX). The interaction of the SP and the SOM (or MENK) effects was studied in cultured locus coeruleus neurons. In neurons loaded with guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), application of SOM (or MENK) evoked a persistent increase in the inward rectifier K+ conductance. A subsequent application of SP suppressed this conductance to a level less than that before the SOM (or MENK) application; the final conductance level was independent of the magnitude of the SOM (or MENK) response. This suppression by SP was persistent, and a subsequent SOM (or MENK) application did not reverse it. When SP was applied to GTP[gamma S]-loaded cells first, subsequent SOM elicited only a small response. In GTP-loaded neurons, application of SP temporarily suppressed the subsequent SOM- (or MENK)-induced conductance increase. These results suggest that the same inward rectifier molecule that responds to an opening signal from GPTX also responds to a closing signal from GnonPTX. The closing signal is stronger than the opening signal.

The role of arachidonic acid metabolism in somatostatin and substance P effects on inward rectifier K conductance in rat brain neurons

Somatostatin enhances an inward rectifier K conductance in cultured locus coeruleus neurons, while substance P reduces an inward rectifier K conductance in cultured nucleus basalis and locus coeruleus neurons. The role of arachidonic acid metabolites in these responses was studied. The somatostatin-induced response was reduced by phospholipase A2 inhibitors, non-specific lipoxygenase inhibitors and specific 5-lipoxygenase inhibitors. A cyclooxygenase inhibitor and a 12-lipoxygenase inhibitor had no effect. 5(S)-HPETE occasionally increased the K conductance, but failed to occlude the somatostatin response. The substance P response was suppressed by a 5-lipoxygenase inhibitor but not by a 12-lipoxygenase inhibitor. These results suggest that the 5-lipoxygenase pathway is not a specific messenger of either one of these responses, but that it plays a more general role in maintaining or enhancing the effectiveness of both somatostatin and substance P responses.

Plastic changes in ara c-treated and “transplanted” coeruleocerebellar cultures

Locus coeruleus axons project to cerebellar cortex in Coeruleocerebellar cultures, where they make functional contacts, and also appear as fine fibers in the outgrowth zones. The predominant catecholamine of locus coeruleus neurons in culture is dopamine. When Coeruleocerebellar cultures are exposed to cytosine arabinoside to destroy cerebellar granule cells and functionally compromise glia, there is a resultant increase of Purkinje cell survival and a sprouting of Purkinje cell recurrent axon collaterals, plus an increase of catecholaminergic axons accompanied by a doubling of tissue dopamine content. If such reorganized cultures are transplanted with granule cells and glia, a second round of plastic changes ensues in which the Purkinje cell population and the recurrent axon collaterals are reduced to control levels, but catecholaminergic axons and dopamine content remain increased. The maintenance of catecholaminergic axons does not appear to depend on the persistence of target neurons.

Differential Actions of Neurotrophins in the Locus Coeruleus and Basal Forebrain

The neurotrophin gene family, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and NT-4/NT-5, supports the survival of distinct peripheral neurons, however, actions upon central neurons are relatively undefined. In this study we have compared different neurotrophins in the regulation of neuronal survival and function using dissociated embryonic cell cultures from two brain regions, the basal forebrain (BF) and locus coeruleus (LC). In the BF, NGF increased choline acetyl transferase (ChAT) activity, but did not influence cholinergic cell survival. In contrast to NGF, BDNF, NT-3, and the novel neurotrophin, NT-4, all increased ChAT activity and cholinergic cell survival. We also examined embryonic LC neurons in culture. LC neurons are unresponsive to NGF. In contrast, NT-3 and NT-4 elicited significant increases in survival of noradrenergic LC neurons, the first demonstration of trophic effects in this critical brain region. Identification of factors supporting coerulcal and basal forebrain neuronal survival may provide insight into mechanisms mediating degeneration of these disparate structures in clinical disorders.

Opioid receptor-mediated inhibition of evoked catecholamine release from cultured neurons of rat ventral mesencephalon and locus coeruleus

The effects of selective opioid agonists on the evoked release of [ 3H]dopamine and [ 3H]noradrenaline were studied in cultured dopaminergic neurons of the ventral mesencephalon (containing the substantia nigra and ventral tegmental area) and in cultured neurons of the noradrenergic locus coeruleus, respectively. The cultures were prepared from embroyonic day 15 rat brains. After 9 days in culture, the calcium-dependent release of [ 3H]dopamine from dopaminergic substantia nigra/ventral tegmental aera neurons induced by 23 mM k + appeared to be inhibited exclusively by activation of κ-opioid receptors, as [ 3H]dopamine release was inhibited selectively by the κ- agonists U69,593 and dynorphin-(1–13) (EC 50 8 and 5 nM, respectively), and this inhibitory effect was antagonized by the κ-selective antagonist nor-binaltorphine (K i 0.07 nM). In contrast, cultured noradrenergic locus coeruleus neurons appeared to contain release-inhibitory μ-opioid receptors only, as evoked [ 3H]noradrenaline release was inhibited selectively by the μ agonist [D-Ala 2, MePhe 4, Gly-ol 5]enkephalin (EC 50 45 nM), a response that was antagonized by the preferential μ antagonist naloxone (K i = 0.7 nM). The δ-opioid receptor agonist [D-Ser 2(O-butyl), Leu 5]enkephaly-Thr 6 did not affect catecholamine release. Dopamine release from cultured ventral mesencephalic neurons, induced by 100 μM N-Methyl-D-Aspartate (NMDA), also appeared to be subject to κ receptor-mediated inhibition, whereas NMDA-induced noradrenaline release from cultured locus coeruleus neurons was under the inhibitor control of μ receptors. It is therefore concluded that in rat brain neurotransmitter release from dopaminergic and noradrenergic neurons, originating from the substantia nigra/vental tegmental area and the locus coeruleus, is liable to inhibition by homogenous populations of κ- and μ-opioid receptors, respectively, independent of the input of non-opioid neurons from distict nuclei.

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.

Locus coeruleus neurons in culture have a developmentally transient α1 adrenergic response

Intracellular recordings from locus coeruleus neurons grown in explant cultures reveal age-related differences in the responses to iontophoretically applied noradrenaline. Cells grown in culture for more than 26 days exhibit a simple hyperpolarizing response while cells cultured for less than 26 days often have biphasic hyperpolarizing-depolarizing responses. Pharmacological studies with selective adrenergic agonists and antagonists show that the hyperpolarizations and hyperpolarizing parts of biphasic responses are mediated by α 2 adrenergic receptors while the depolarizing responses are mediated by α 1 receptors. These observations may reflect a population of neurotransmitter receptors which is transiently present during early postnatal development.

Locus coeruleus neurons in culture have a developmentally transient alpha 1 adrenergic response.

Intracellular recordings from locus coeruleus neurons grown in explant cultures reveal age-related differences in the responses to iontophoretically applied noradrenaline. Cells grown in culture for more than 26 days exhibit a simple hyperpolarizing response while cells cultured for less than 26 days often have biphasic hyperpolarizing-depolarizing responses. Pharmacological studies with selective adrenergic agonists and antagonists show that the hyperpolarizations and hyperpolarizing parts of biphasic responses are mediated by alpha 2 adrenergic receptors while the depolarizing responses are mediated by alpha 1 receptors. These observations may reflect a population of neurotransmitter receptors which is transiently present during early postnatal development.

Hyperpolarizing and age-dependent depolarizing responses of cultured locus coeruleus neurons to noradrenaline

The electrical activity and responses to noradrenaline (NA) of locus coeruleus (LC) neurons have been studied in organotypic cultures using intracellular recording. Most LC neurons were predominantly quiescent, though occasional bursts of activity were observed; a few cells were tonically active at rates of 0.5–5/s. In most cells tested, iontophoretic application of NA evoked responses which were initially hyperpolarizing, sometimes followed by a depolarizing phase and frequently followed by a period of increased excitatory synaptic activity. The enhanced synaptic activity appeared to be an indirect effect since it was blocked by bath application of tetrodotoxin (TTX). In the presence of TTX, responses to NA of all but one cell were simple hyperpolarizations or biphasic (hyperpolarization/depolarization) responses. The presence of the depolarizing component appeared to be age-dependent, since it was frequently observed in cultures grown in vitro for less than 26 days, while neurons in older cultures exhibited only hyperpolarizing responses. If such age-dependent depolarizing responses are present in vivo, they would represent a unique example of a transmitter response which is present only during a transient developmental phase.

Noradrenergic actions of purkinje and locus coeruleus neurons in culture

1. Explant cultures prepared from neonatal mice were used to study the actions of ionto-phoretically applied noradrenaline (NA) on Purkinje and locus coeruleus neurons, with extracellular and intracellular recording respectively. 2. NA depressed spontaneous activity of Purkinje neurons and enhanced excitatory responses to glutamate in 14/16 cells. 3. In cultures older than 26 days, NA hyperpolarized LC neurons but had no effect on or depressed depolarizing responses to glutamate. The hyperpolarizations were blocked by the selective α 2 antagonist yohimbine. 4. Enhancement of glutamate responses is not a ubiquitous characteristic of NA action and appears not to be associated with α 2 adrenergic receptors.

Response of single motoneurons to direct stimulation in toad's spinal cord.

Response of single motoneurons to direct stimulation in toad's spinal cord.