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Notes: Abstract: One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of 32PO4 into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time-dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one-trial conditioning.

Notes: Abstract: It is well established that ischemia-induced release of glutamate and the subsequent activation of postsynaptic glutamate receptors are important processes involved in the development of ischemic neuronal damage. Moderate intraischemic hypothermia attenuates glutamate release and confers protection from ischemic damage, whereas mild intraischemic hyperthermia increases glutamate release and augments ischemic pathology. As protein kinase C (PKC) is implicated in neurotransmitter release and glutamate receptor-mediated events, we evaluated the relationship between intraischemic brain temperature and PKC activity in brain regions known to be vulnerable or nonvulnerable to transient global ischemia. Twenty minutes of bilateral carotid artery occlusion plus hypotension were induced in rats in which intraischemic brain temperature was maintained at 30°C, 37°C, or 39°C. Prior to and following ischemia, brain temperature was 37°C in all groups. Cytosolic, membrane-bound, and total PKC activities were determined in hippocampal, striatal, cortical, and thalamic homogenates at the end of ischemia and at 0.25–24 h of recirculation. PKC activity of control rats varied by region and were affected by altered brain temperature. For both membrane-bound and cytosolic PKC, there was a significant temperature effect, and for membrane-bound PKC there was also a significant effect of region. Rats with normothermic ischemia (37°C) showed extensive depressions of all PKC fractions. Hippocampus and striatum were noteworthy for depressions in PKC activity extending from the earliest (15 min) to the latest (24 h) recirculation times studied, whereas cortex showed PKC depressions chiefly during the first hour of recirculation, and the thalamic pattern was inconsistent. In contrast, in rats with hypothermic ischemia (30°C), significant overall effects were noted only for total PKC in thalamus, which showed depressed levels at both 1 and 24 h of recirculation. Rats with hyperthermic (39°C) ischemia also showed significant overall effects for the time course of membrane-bound, cytosolic, and total PKC activities in the hippocampus, striatum, and cortex. However, no significant reductions in PKC indices were observed in the thalamus. For membrane-bound PKC, significant temperature effects were noted for hippocampus, striatum, and cortex, but not for thalamus. For cytosolic, as well as total PKC, activity, significant temperature effects were noted for all four brain regions. Our results indicate that ischemia, followed by reperfusion, induces a significant reduction in PKC activity and that this process is highly influenced by the brain temperature during ischemia. Furthermore, our data also establish that differences exist in the response of PKC to ischemia/recirculation in vulnerable versus non-vulnerable brain regions. These results suggest that PKC alterations may be an important factor involved in the modulatory effects of temperature on the outcome following transient global ischemia.

Notes: Abstract: We investigated the role of Ca2+-dependent protein kinases in the regulation of astrocytic cell volume. Calmodulin (CaM) antagonists were used to inhibit CaM and thus Ca2+/CaM-dependent protein kinase. The effect of these inhibitors as well as activators and inhibitors of protein kinase C (PKC) on astrocytic volume was measured in response to hypoosmotic stress and under isoosmotic conditions. In conditions of hypoosmolarity, CaM antagonists had no effect on swelling, but inhibited the regulatory volume decrease. PKC activation facilitated the swelling induced by hypoosmotic stress. PKC inhibitors induced cell shrinkage and inhibited the initial phase of regulatory volume decrease, whereas PKC down-regulation caused pronounced swelling and partial inhibition of regulatory volume decrease. In isoosmotic conditions, CaM antagonists and PKC activation did not affect astrocytic volume, but PKC inhibitors caused shrinking and PKC down-regulation led to swelling of these cells. These studies indicate the importance of Ca2+-dependent protein kinases in the regulation of astrocytic cell volume.

Notes: The mitogen-activated protein kinase (MAPK) cascade is an important contributor to synaptic plasticity and learning in both vertebrates and invertebrates. In the nudibranch mollusk Hermissenda, phosphorylation and activation of the extracellular signal-regulated protein kinase (ERK), a key member of a MAPK cascade, is produced by one-trial and multitrial Pavlovian conditioning. Several signal transduction pathways that are activated by 5-hydroxytryptamine (5-HT) and may contribute to conditioning have been identified in type B photoreceptors. However, the regulation of ERK activity by ‘upstream’ signaling molecules has not been previously investigated in Hermissenda. In the present study we examined the role of protein kinase C (PKC) in the serotonin (5-HT) activation of the ERK pathway. The phorbol ester TPA produced an increase in ERK phosphorylation that was blocked by the PKC inhibitors GF109203X or Gö6976. TPA-dependent ERK phosphorylation was also blocked by the MEK1 inhibitors PD098059 or U0126. The increased phosphorylation of ERK by 5-HT was reduced but not blocked by pretreatment with the calcium chelator BAPTA-AM or pretreatment with Gö6976 or GF109203X. These results indicate that Ca2+-dependent PKC activation contributes to ERK phosphorylation, although a PKC-independent pathway is also involved in 5-HT-dependent ERK phosphorylation and activation.

Notes: ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKs. Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRs.

Notes: Neuroinflammation is associated with a variety of CNS pathologies. Levels of tumor necrosis factor-alpha (TNF-α), a major proinflammatory cytokine, as well as extracellular ATP, are increased following various CNS insults. Here we report on the relationship between ATP/P2 purinergic receptor activation and lipopolysaccharide (LPS)-induced TNF-α release from primary cultures of rat cortical astrocytes. Using ELISA, we confirmed that treatment with LPS stimulated the release of TNF-α in a concentration and time dependent manner. ATP treatment alone had no effect on TNF-α release. LPS-induced TNF-α release was attenuated by 1 mm ATP, a concentration known to activate P2X7 receptors. Consistent with this, 3′-O-(4-Benzoyl)benzoyl-ATP (BzATP), a P2X7 receptor agonist, also attenuated LPS-induced TNF-α release. This reduction in TNF-α release was not due to loss of cell viability. Adenosine and 2-chloroadenosine were ineffective, suggesting that attenuation of LPS-induced TNF-α release by ATP was not due to ATP breakdown and subsequent activation of adenosine/P1 receptors. Interestingly, treatment of astrocyte cultures with 10 µm or 100 µm ATP potentiated TNF-α release induced by a submaximal concentration of LPS. UTP and 2methylthioADP (2-MeSADP), P2Y receptor agonists, also enhanced this LPS-induced TNF-α release. Our observations demonstrate opposing effects of ATP/P2 receptor activation on TNF-α release, i.e. P2X receptor activation attenuates, whereas P2Y receptor activation potentiates TNF-α release in LPS-stimulated astrocytes. These observations suggest a mechanism whereby astrocytes can sense the severity of damage in the CNS via ATP release from damaged cells and can modulate the TNF-α mediated inflammatory response depending on the extracellular ATP concentration and corresponding type of astrocyte ATP/P2 receptor activated.

Notes: Mechanical or ischemic trauma to the CNS causes the release of nucleotides and other neurotransmitters into the extracellular space. Nucleotides can activate nucleotide receptors that modulate the expression of genes implicated in cellular adaptive responses. In this investigation, we used human 1321N1 astrocytoma cells expressing a recombinant P2Y2 receptor to assess the role of this receptor in the regulation of anti-apoptotic (bcl-2 and bcl-xl) and pro-apoptotic (bax) gene expression. Acute treatment with the P2Y2 receptor agonist UTP up-regulated bcl-2 and bcl-xl, and down-regulated bax, gene expression. Activation of P2Y2 receptors was also coupled to the phosphorylation of cyclic AMP responsive element binding protein that positively regulates bcl-2 and bcl-xl gene expression. Cyclic AMP responsive element decoy oligonucleotides markedly attenuated the UTP-induced increase in bcl-2 and bcl-xl mRNA levels. Activation of P2Y2 receptors induced the phosphorylation of the pro-apoptotic factor Bad and caused a reduction in bax/bcl-2 mRNA expression ratio. All these signaling pathways are known to be involved in cell survival mechanisms. Using cDNA microarray analysis and RT–PCR, P2Y2 receptors were found to up-regulate the expression of genes for neurotrophins, neuropeptides and growth factors including nerve growth factor 2; neurotrophin 3; glia-derived neurite-promoting factor, as well as extracellular matrix proteins CD44 and fibronectin precursor – genes known to regulate neuroprotection. Consistent with this observation, conditioned media from UTP-treated 1321N1 cells expressing P2Y2 receptors stimulated the outgrowth of neurites in PC-12 cells. Taken together, our results suggest an important novel role for the P2Y2 receptor in survival and neuroprotective mechanisms under pathological conditions.

Notes: The present study has been aimed at characterizing the ATP/P2 receptor (and transductional pathways) responsible for the morphological changes induced in vitro by αβmethyleneATP on rat astrocytes obtained from cerebral cortex, a brain area highly involved in neurodegenerative diseases. Exposure of cells to this purine analogue resulted in elongation of cellular processes, an event reproducing in vitro a major hallmark of in vivo reactive gliosis. αβmethyleneATP-induced gliosis was prevented by the P2X/P2Y blocker pyridoxalphosphate-6-azophenyl-2′-4′-disulfonic acid, but not by the selective P2X antagonist 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, ruling out a role for ligand-gated P2X receptors. Conversely, the Gi/Go protein inactivator pertussis toxin completely prevented αβmethyleneATP-induced effects. No effects were induced by αβmethyleneATP on intracellular calcium concentrations. RT-PCR and western blot analysis showed that αβmethyleneATP–induced gliosis involves up-regulation of cyclooxygenase-2 (but not lipooxygenase). Also this effect was fully prevented by pyridoxalphosphate-6-azophenyl-2′-4′-disulfonic acid. Experiments with inhibitors of mitogen-activated protein kinases (MAPK) suggest that extracellular signal regulated protein kinases (ERK)1/2 mediate both cyclooxygenase-2 induction and the associated in vitro gliosis. These findings suggest that purine-induced gliosis involves the activation of a calcium-independent G-protein-coupled P2Y receptor linked to ERK1/2 and cyclooxygenase-2. Based on the involvement of cyclooxygenase-2 and inflammation in neurodegenerative diseases, these findings open up new avenues in the identification of novel biological targets for the pharmacological manipulation of neurodegeneration.

Notes: [Auszug] Hermissenda were trained by the following conditioning procedure8. First, the times taken (latency) by individual animals to enter an illuminated area were measured to establish individual baselines, and the animals were then trained in an automated apparatus14 by a protocol involving stimulation ...

Notes: Summary [3H]Agmatine (amino-4-guanidobutane) has been shown to be potentially useful for identifying and assessing the ACh sensitivity of specific neurons. Small cationic amines are able to permeate ACh-activated ion channels in sympathetic neurons and vertebrate endplates. Sensory neurons of the photic pathway in the nudibranch molluscHermissenda crassicornis are cholinergic and the synaptic interactions between the photic and vestibular systems have been well characterized electrophysiologically. We have therefore tested the feasibility of using autoradiography with [3H]agmatine, (a) to identify known ACh-responsive postsynaptic cells and (b) to examine its ability to serve as an indicator of physiologic activity within the photic and vestibular pathways under conditions of darkness and light stimulation. Scintillation counting revealed that ∼ 70% of the radioactivity was associated with the CNS while ∼ 30% was found in the processing fluids, indicating that routine glutaraldehyde-osmium fixation and subsequent processing for epoxy embedding allows retention of substantial amounts of the radiolabel. The autoradiographic results consistently demonstrated that the uptake patterns for [3H]agmatine did reflect some of the known neuronal interactions under the experimental conditions of light and dark. The accuracy extended to the second order cells of the optic ganglion and to putative interneurons along the photic tract in the cerebropleural ganglion. Since all the neurons in these pathways are unipolar with their synaptic interactions occurring only at the terminal endings, the radiolabel accumulated in the somata resulted from retrograde axonal transport. In the photic-vestibular pathways, the highest silver grain densities were found over structures (cell bodies or axon tracts) with increased synaptic activity coupled with higher levels of cellular activity (i.e. increased excitatory postsynaptic potentials or increased spontaneous impulse activity). Slightly less label was found in cells which received increased numbers of inhibitory postsynaptic potentials that produced hyperpolarization and a transient cessation of impulse activity under conditions of illumination. Therefore, the uptake levels of [3H]agmatine as revealed by autoradiography appear to reflect not only changes in sensitivity or density of ACh-activated channels but also changes in cellular activity as indicated by increased amounts of retrograde transport. These results represent the first example of the effective use of this radiolabel as an indicator of synaptic activity in invertebrates and in sensory systems.