Cultured Locus Coeruleus Research Articles

Neurotoxin-induced DNA damage is persistent in SH-SY5Y cells and LC neurons.

Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathological characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxin camptothecin (CPT) to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured locus coeruleus (LC) and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficient in DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathological characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insults in vivo.

BMPs, FGF8 and Wnts regulate the differentiation of locus coeruleus noradrenergic neuronal precursors

In the present study, we investigated the involvement of rhombomere 1 patterning proteins in the regulation of the major noradrenergic centre of the brain, the locus coeruleus. Primary cultures of rat embryonic day 13.5 locus coeruleus were treated with fibroblast growth factor-8, noggin and members of the bone morphogenetic and Wnt protein families. We show that bone morphogenetic proteins 2, 5 and 7 increase and noggin decreases the number of tyrosine hydroxylase-positive locus coeruleus neurons. Interestingly, from all Wnts expressed in the first rhombomere by embryonic day 12.5 in the mice, we only found expression of wnt5a mRNA in the vicinity of the locus coeruleus. In agreement with this finding, from all Wnts studied in vitro, only Wnt5a increased the number of tyrosine hydroxylase-positive neurons in locus coeruleus cultures. Finally, we also found that fibroblast growth factor-8 increased the number of tyrosine hydroxylase-positive cells in locus coeruleus cultures. Neither of the identified factors affected the survival of tyrosine hydroxylase-positive locus coeruleus noradrenergic neurons or the proliferation of their progenitors or neurogenesis. Instead, our results suggest that these patterning signals of rhombomere 1 may work to promote the differentiation of noradrenergic progenitors at later stages of development.

Orexin (hypocretin) effects on constitutively active inward rectifier K+ channels in cultured nucleus basalis neurons.

Orexins are excitatory transmitters implicated in sleep disorders. Because orexins were discovered only recently, their ionic and signal transduction mechanisms have not been well clarified. We recently reported that orexin A (OXA) inhibits G protein-coupled inward rectifier K+ (GIRK) channels in cultured locus coeruleus and nucleus tuberomammillaris neurons. Other work in our laboratory revealed the existence of a novel inward rectifier K+ channel (KirNB), which is located in cholinergic neurons of the nucleus basalis (NB) and possesses unique single-channel characteristics. The mean open time is considerably shorter in KirNB than in Kir2.0 channels. Constitutive activity and a smaller unitary conductance set KirNB apart from cloned Kir3.0 channels. Previously, we found that substance P excites NB neurons by inhibiting KirNB channels. Here we show that orexins suppress KirNB channel activity, likely leading to neuronal excitation. Electrophysiological studies were performed on cultured NB neurons from the basal forebrain. OXA application decreased whole cell conductance through a pertussis toxin (PTX)-insensitive G protein. The OXA-suppressed current was inwardly rectifying with a reversal potential around E(K). Single-channel recordings of NB neurons revealed that constitutively active KirNB channels were transiently inhibited by OXA. Okadaic acid pretreatment abolished the recovery. The results suggest that OXA inhibition of KirNB is mediated by a PTX-insensitive G protein (i.e., G(q/11)), which eventually results in channel phosphorylation. Recovery from this inhibition is by dephosphorylation. These results, taken together with our previous study, suggest that orexin receptors can elicit neuronal excitation through at least two families of inward rectifier K+ channels: GIRK and KirNB channels.

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.

Different G proteins mediate somatostatin-induced inward rectifier K+ currents in murine brain and endocrine cells.

1. Types of G proteins (G protein alpha-subunit subtypes) which mediate the activation of inward rectifier K+ currents by somatostatin (somatotrophin release-inhibiting factor, SRIF) were determined in cultured locus coeruleus neurones from newborn rats and in AtT-20 cells (a mouse pituitary cell line). 2. The whole-cell patch clamp technique was used together with injection of antibodies against pertussis toxin (PTX)-sensitive G protein alpha-subunits or with injection of antisense (or sense) oligonucleotides against these G proteins. 3. In locus coeruleus neurones, the SRIF-induced activation of inward rectifier K+ currents was inhibited by anti-G alpha i1/G alpha i2 antibody injection, but not by anti-G alpha i3 or by anti-G alpha o/G alpha i3 antibody injection, suggesting that the SRIF response is mediated through G alpha i1 and/or G alpha i2. 4. The SRIF-induced activation of the inward rectifier was suppressed in locus coeruleus neurones after injection of antisense oligonucleotides against G alpha i2, but not by injection of sense oligonucleotides against G alpha i2. Injection of antisense (or sense) oligonucleotides against G alpha i1, G alpha i3 and G alpha O (common) had no effect. These results suggest that G alpha i2 is involved in this SRIF response. 5. In AtT-20 cells, the SRIF-induced activation of inward rectifier K+ currents was suppressed by injection of anti-G alpha i3 antibody, but not by injection of anti-G alpha i1/G alpha i2 antibody. 6. The above results indicate that Gi mediates the SRIF effects on inward rectifier K+ currents. However, different subtypes of Gi are involved in the brain neurones and in the endocrine cells: Gi2 in locus coeruleus neurones and Gi3 in AtT-20 cells.

Opposing effects of morphine and the neurotrophins, NT-3, NT-4, and BDNF, on locus coeruleus neurons in vitro

The development of noradrenergic locus coeruleus (LC) neurons is subject to regulation by multiple epigenetic signals. To examine the potential regulation of LC ontogeny by opiates and neurotrophins, we studied the effects of morphine and NT-3, NT-4, and BDNF on the survival and differentiation of LC neurons from prenatal rats in dissociated cell culture. Noradrenergic cells were identified and counted following tyrosine hydroxylase (TH) immunocytochemistry, and their state of differentiation was assessed by measuring norepinephrine (NE) uptake. Treating LC cultures with morphine starting on day 1 after plating resulted in a 20% decrease in NE uptake and a small (12%) but significant decrease in the number of TH-immunoreactive (TH + ) cells. Application of morphine on day 4 after plating had the same effect on NE uptake without influencing TH + cell number. This effect of morphine was blocked by concomitant exposure to naloxone (an opioid receptor antagonist), and mimicked by exposure to opioid peptides. Treatment of cultures with the neurotrophins, NT-3 or NT-4, increased NE uptake and TH + cell number, as reported previously. Moreover, we show for the first time that brain-derived neurotrophic factor (BDNF) exerts similar effects, with a large (110%) increase in NE uptake and a modest (20%) increase in TH + cell number. Cotreatment of LC cultures with morphine and NT-3 resulted in an attenuation of the NT-3 effect on both NE uptake and the number of TH + cells. In contrast, cotreatment of LC cultures with morphine and NT-4 or BDNF attenuated the neurotrophin effect on TH + cell number but not on NE uptake. Our results raise the possibility that opioid peptides may modulate the influence of neurotrophins on LC neuronal survival and differentiation.

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.

Neurotrophic effects of cAMP generating systems on central noradrenergic neurons

The environmental signals which regulate the development of central noradrenergic neurons are largely unknown. The aim of the present study was to search for factors affecting the development of these cells. Dissociated cultures of embryonic dorsal brainstem tissue, containing the nucleus locus coeruleus (LC), were established; norepinephrine (NE) and GABA uptake were assessed, and noradrenergic versus total neurons were identified and counted following immunocytochemical staining with tyrosine hydroxylase (TH) and neuron specific enolase (NSE) antibodies, respectively. Application of dibutyryl cAMP (dbcAMP), other cAMP analogs, or forskolin, to LC cultures resulted in a significant increase in NE uptake which was associated with up to a 4-fold increase in the number of TH immunoreactive cells (TH+). dbcAMP treatment caused an increase in the number of TH+ cells in LC cultures by enhancing their survival and/or by upregulating their phenotypic differentiation. A possible effect of dbcAMP on cell proliferation and transformation of non-noradrenergic cells to noradrenergic TH+ cells were examined and suggested not to underlie this effect of cAMP. Glial cells may mediate the effect of cAMP on noradrenergic neurons. Calcium was not involved in the trophic activity of dbcAMP, which was probably mediated by protein phosphorylation via cAMP dependent protein kinase. Insulin (25 μg/ml) was found to increase the number of TH+ cells by 73%. The β-adrenergic agonist isoproterenol also increased the number of TH+ cells by 53%. We propose a neurotrophic role for NE during development of central noradrenergic neurons.

Convergent regulation by ciliary neurotrophic factor and dopamine of tyrosine hydroxylase expression in cultures of rat substantia nigra

Ciliary neurotrophic factor and dopamine were found to enhance the expression of tyrosine hydroxylase immunoreactivity in cultured neurons from the substantia nigra of 16-day-old rat fetuses. The number of tyrosine hydroxylase-positive cells decreased progressively to ~ 30% by 96 h. Treatment with 5 μM dopamine maintained the tyrosine hydroxylase-positive neurons at 60% for 48 h, but not for longer. Concurrent treatment with 5 μM dopamine and 20 trophic units/ml ciliary neurotrophic factor had a greater impact on tyrosine hydroxylase-positive cells, resulting in the maintenance of 70% of the initial number for up to 72 h, but not beyond that time. When dopamine or dopamine/ciliary neurotrophic factor treatments were applied for 24 h after a 48-h delay, the number of tyrosine hydroxylase-positive cells was restored to 60 and 80%, respectively, but no restoration was observed with 96-h delayed treatments. These results suggest that dopamine and ciliary neurotrophic factor, alone or in combination, are not able to support the survival of tyrosine hydroxylase-positive neurons, but reduce their apparent numerical loss by enhancing the expression of tyrosine hydroxylase. The effects of dopamine, alone or in combination with ciliary neurotrophic factor, were predominantly mediated by D 2 receptors, since they were blocked by selective D 2 receptor antagonists and since the D 2 receptor agonist quinpirole was able to substitute for dopamine. The effects of dopamine and ciliary neurotrophic factor were similar in astroblast-rich and in astroblast-depleted cultures, suggesting that they were not mediated through glial cells. These results extend our previous observations on locus coeruleus cultures, in which the concurrent treatment with ciliary neurotrophic factor and norepinephrine was shown to enhance tyrosine hydroxylase expression (but not survival) of noradrenergic neurons. They also consolidate the view that ciliary neurotrophic factor and the neuron's own transmitter act in convergence and in an autocrine/paracrine mode as regulators of the corresponding neurotransmitter phenotype.

Cooperative Effects of Ciliary Neurotrophic Factor and Norepinephrine on Tyrosine Hydroxylase Expression in Cultured Rat Locus Coeruleus Neurons

Ciliary neurotrophic factor (CNTF) was found to promote the expression of tyrosine hydroxylase (TH) immunoreactivity by cultured noradrenergic neurons from the locus coeruleus (LC) of E18 rat fetuses, but only in the concomitant presence of norepinephrine (NE), their own neurotransmitter. The number of TH-positive cells in LC cultures was shown to decrease by 65% within 3 days and by 75% after 6 days. Treatment with 10 TU/ml human recombinant CNTF together with 1 μ M NE was able to fully maintain the initial number of TH-positive neurons for 3 days. This effect, however, was no longer seen after 6 days of continuous exposure. A 24-hr treatment with CNTF/NE was capable of completely restoring the initial number of TH-positive cells, even if its addition was delayed for 2 days. Moreover, when its addition was delayed for 5 days, CNTF/NE restored ∼80% of the TH-positive neurons that were initially present. These results suggest that the disappearance of TH-positive neurons in LC cultures is not due to their death, but rather to the reduced expression of TH and that the simultaneous exposure to CNTF and NE upregulates TH. Effects on TH-positive cell number were not evoked by CNTF or NE alone. The CNTF/NE effect was dependent on protein synthesis, but was only partially inhibited by RNA synthesis inhibitors, suggesting that both transcription from preexisting mRNA and synthesis of new RNA were stimulated. The effect of CNTF/NE was mediated by α 2-adrenoceptors, since it was blocked by α 2-antagonists and since α 2-agonists were able to substitute for NE. Our results suggest a novel mechanism of regulation of the phenotype of the noradrenergic LC neuron, involving the collaborative influences of CNTF and norepinephrine, their own neurotransmitter.

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.

Chapter 43 Regulation of the development of locus coeruleus neurons in vitro

Primary dissociated cultures of embryonic locus coeruleus (LC) neurons were established. Norepinephrine (NE) uptake was used as an index of maturation of the noradrenergic (NA) neurons from the LC. When LC cells were cocultured with a low density of hippocampal target cells, NE uptake was stimulated. However, increasing the concentration of hippocampal cells resulted in a significant decrease in NE uptake. The target stimulatory effect was mediated by both neurons and glia, whereas the inhibitory effect was mediated by direct contact between target glia and LC neurons and detected only in the presence of serum. In addition to the effect of target, we also tested the effect of elevated intracellular cAMP levels on NE uptake versus GABA uptake. GABA uptake was an index of development of non-NA cells. Increasing intracellular cAMP resulted in selective stimulation of NE uptake. These studies illustrate the potential of dissociated LC cultures in studying the regulation of NA axonal outgrowth.

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

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

Somatostatin induces an inward rectification in rat locus coeruleus neurones through a pertussis toxin‐sensitive mechanism.

1. Membrane properties and somatostatin effects were studied in cultured locus coeruleus neurones from neonatal rats by using the whole-cell version of the patch clamp technique. 2. The current-voltage relationship of the resting cell revealed an inward-going rectification. The inward currents developed almost instantaneously upon hyperpolarizing the membrane under voltage clamp, and at large negative potentials the inward current showed a time-dependent inactivation. Extracellularly applied Cs+ or Ba2+ (0.1 mM) inhibited the inward current in a voltage-dependent manner. 3. Application of somatostatin (0.01-1 microM) produced an increase in membrane conductance. Somatostatin-induced currents were calculated by subtracting the control current from the current during the somatostatin-induced response. The somatostatin-induced current developed almost instantaneously with hyperpolarization and did not show any time-dependent inactivation. The current-voltage relationship of the somatostatin-induced current exhibited a rectification in the inward direction and showed a reversal potential. The reversal potentials were close to the K+ equilibrium potential. 4. Extracellular Cs+ or Ba2+ (0.1 mM) inhibited the somatostatin-induced currents in a voltage-dependent manner, the effectiveness increasing with hyperpolarization. The somatostatin-induced hyperpolarization was not affected by apamin (20 nM) or by charybdotoxin (100 nM). 5. These results indicate that the somatostatin-induced conductance is very similar to the inward-rectification conductance. Because the somatostatin-induced inward rectification did not exhibit a time-dependent inactivation, this rectification and the inward rectification in the control neurones may arise from two different channels. 6. Pre-treatment of neurones with pertussis toxin abolished the somatostatin-induced response, but did not affect the resting inward rectification. When GTP gamma S was applied intracellularly, somatostatin produced an irreversible activation of the inward rectification conductance. The somatostatin-induced hyperpolarization may therefore be mediated through a pertussis toxin-sensitive GTP-binding protein.

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.

Cerebellum plus locus coeruleus in tissue culture. II: Development and metabolism of catecholamines.

In contrast to locus coeruleus neurons in vivo, dopamine was the predominant catecholamine synthesized, stored, and released by neonatal mouse locus coeruleus cultures which included target cerebellar tissue, and norepinephrine was present in these cultures only at very low levels. Developmentally, norepinephrine increased slightly in the explants during the first 4 days in vitro and declined thereafter to barely detectable levels, whereas dopamine began to rise after 4 days and reached maximal levels by 7 days. Dopamine beta-hydroxylase was present in these cultures throughout maturation. These results suggest that the high ratio of dopamine to norepinephrine in locus coeruleus cultures cannot be attributed to the absence of appropriate target tissue or to a lack of the enzyme, dopamine beta-hydroxylase.

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.

Viability of locus coeruleus cultures in the fourth ventricle of the adult rat

Both tyrosine hydroxylase and dopamine-beta-hydroxylase immunocytochemistry indicated the survival of catecholamine neurons and processes in 7-day-old organotype cultures of the locus coeruleus. Cultures of this age were transplanted in the 4th ventricle of adult rats via the cisterna magna. Two months after transplantation microscopic observations revealed that the transplant developed with a defined neural organization. Following tyrosine hydroxylase immunocytochemistry, immunoreactive cells were identified within the transplants. These results indicate that organotype cultures of the locus coeruleus survive within the 4th ventricle of adult rats.

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

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