The Australian EEG database.

The Australian EEG Database is a web-based de-identified searchable database of 18,500 EEG records recorded at a regional public hospital over an 11-year period. Patients range in age from a premature infant born at 24 weeks gestation, through to people aged over 90 years. This paper will describe the history of the database, the range of patients represented in the database, and the nature of the text-based and digital data contained in the database. Preliminary results of the first two studies undertaken using the database are presented. Plans for sharing data from the Australian EEG database with researchers are discussed. We anticipate that such data will be useful in not only helping to answer clinical questions but also in the field of mathematical modeling of the EEG.

Autonomous activity of CaMKII is only transiently increased following the induction of long-term potentiation in the rat hippocampus.

A major role has been postulated for a maintained increase in the autonomous activity of CaMKII in the expression of long-term potentiation (LTP). However, attempts to inhibit the expression of LTP with CaMKII inhibitors have yielded inconsistent results. Here we compare the changes in CaMKII autonomous activity and phosphorylation at Thr286 of alphaCaMKII in rat hippocampal slices using chemical or tetanic stimulation to produce either LTP or short-term potentiation (STP). Tetanus-induced LTP in area CA1 requires CaMKII activation and Thr286 phosphorylation of alphaCaMKII, but we did not observe an increase in autonomous activity. Next we induced LTP by 10 min exposure to 25 mM tetraethyl-ammonium (TEA) or 5 min exposure to 41 mM potassium (K) after pretreatment with calyculin A. Exposure to K alone produced STP. These protocols allowed us to monitor temporal changes in autonomous activity during and after exposure to the potentiating chemical stimulus. In chemically induced LTP, autonomous activity was maximally increased within 30 s whereas this increase was significantly delayed in STP. However, in both LTP and STP the two-fold increase in autonomous activity measured immediately after stimulation was short-lived, returning to baseline within 2-5 min after re-exposure to normal ACSF. In LTP, but not in STP, the phosphorylation of alphaCaMKII at Thr286 persisted for at least 60 min after stimulation. These results confirm that LTP is associated with a maintained increase in autophosphorylation at Thr286 but indicate that a persistent increase in the autonomous activity of CaMKII is not required for the expression of LTP.

Angiotensin II promotes the phosphorylation of cyclic AMP-responsive element binding protein (CREB) at Ser133 through an ERK1/2-dependent mechanism.

In cells from the adrenal medulla, angiotensin II (AII) regulates both the activity and mRNA levels of catecholamine biosynthetic enzymes whose expression is thought to be under the control of cAMP-responsive element (CRE) binding protein (CREB). In this study, we evaluated the effect of AII stimulation on CREB phosphorylation at Ser133 (pCREB) in bovine adrenal chromaffin cells (BACC). We found that AII produces a rapid and AII type-1 receptor (AT1)-dependent increase in pCREB levels, which is blocked by the MEK1/2 inhibitor U0126 but not by H-89, SB203580 or KN-93, suggesting that it is mediated by the extracellular-regulated protein kinases 1 and 2 (ERK1/2) and not by cAMP-dependent protein kinase (PKA), p38 mitogen-activated protein kinase (p38MAPK) or Ca(2+)/calmodulin-dependent protein kinases (CaMKs) dependent pathways. Gel-shift experiments showed that the increase in pCREB levels is accompanied by an ERK1/2-dependent upregulation of CRE-binding activity. We also found that AII promotes a rapid and reversible increase in the activity of the non-receptor tyrosine kinase Src and that the inhibition of this enzyme completely blocks the AII-induced phosphorylation of ERK1/2, the CREB kinase (p90)RSK and CREB. Our data support the hypothesis that in BACC, AII upregulates CREB functionality through a mechanism that requires Src-mediated activation of ERK 1/2 and (p90)RSK.

Autonomous activity and autophosphorylation of CAMPK-II in rat hippocampal slices: effects of tissue preparation.

Measurement of the proportion of calcium/calmodulin-stimulated protein kinase II (CaMPK-II) that is autonomously active or phosphorylated on Thr(286) is thought to provide an index of the degree to which CaMPK-II in a tissue has been activated. We have examined how various ways of handling hippocampal tissue can alter these properties. Both autonomous activity and phospho-Thr(286) content was high in freshly dissected hippocampus or freshly cut hippocampal slices. After incubation of hippocampal slices in artificial cerebrospinal fluid for 120 min, both properties of CaMPK-II decreased to a steady state level. Freeze-thaw or cutting the equilibrated slices could rapidly increase both autonomous activity and phospho-Thr(286) immunoreactivity of CaMPK-II. These increases were comparable to changes induced by experimental treatment. Therefore, our results suggest that considerable care needs to be taken over the way in which hippocampal slices are handled.

Modulation of the phosphorylation and activity of calcium/calmodulin-dependent protein kinase II by zinc.

Calcium/calmodulin-dependent protein kinase II (CaMPK-II) is a key regulatory enzyme in living cells. Modulation of its activity, therefore, could have a major impact on many cellular processes. We found that Zn(2+) has multiple functional effects on CaMPK-II. Zn(2+) generated a Ca(2+)/CaM-independent activity that correlated with the autophosphorylation of Thr(286), inhibited Ca(2+)/CaM binding that correlated with the autophosphorylation of Thr(306), and inhibited CaMPK-II activity at high concentrations that correlated with the autophosphorylation of Ser(279). The relative level of autophosphorylation of these three sites was dependent on the concentration of zinc used. The autophosphorylation of at least these three sites, together with Zn(2+) binding, generated an increased mobility form of CaMPK-II on sodium dodecyl sulfate gels. Overall, autophosphorylation induced by Zn(2+) converts CaMPK-II into a different form than the binding of Ca(2+)/CaM. In certain nerve terminals, where Zn(2+) has been shown to play a neuromodulatory role and is present in high concentrations, Zn(2+) may turn CaMPK-II into a form that would be unable to respond to calcium signals.

Phosphorylation of a new brain-specific septin, G-septin, by cGMP-dependent protein kinase.

The septins are a family of GTPase enzymes, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. We purified and cloned the cDNA for a 40-kDa protein from rat brain that is a substrate for type I cGMP-dependent protein kinase (PKG). The amino acid sequences of two tryptic peptides of P40 showed high homology to the septins. Molecular cloning revealed the 358-amino acid P40 to be a new member of the septin family. P40 was named G-septin, as it is phosphorylated in vitro by PKG, but relatively poorly by the related cAMP-dependent protein kinase and not by protein kinase C. Two splice variants of G-septin (alpha and beta) were found with distinct N and C termini, but a common GTPase domain. G-septin lacks the C-terminal coiled-coil domain characteristic of all other mammalian septins and uniquely has two predicted phosphorylation site motifs for type I PKG. Photoaffinity labeling with [alpha-(32)P]GTP confirmed that G-septin is a GTP-binding protein. Northern blotting showed that G-septin mRNA (5.0 kilobases) is highly expressed in brain and undetectable in 12 other tissues, indicating that the G-septins are primarily neuronal proteins. Very low levels of 6.0-, 3.4-, and 2.6-kilobase transcripts were found in testis. Our results reveal a new class of brain-specific septins that may be regulated by PKG in neurons.

Inhibition of transmitter release and long-term depression in the avian hippocampus.

Long-term depression has recently been shown to occur at glutamatergic synapses in the avian hippocampus and requires activation of calcium/calmodulin-dependent protein kinase II in the nerve terminal. Here using whole cell and intracellular recordings from brain slices, we show that the N-type calcium channel contributes significantly to glutamate release in the avian hippocampus. Activation of the metabotrobic gamma-aminobutyric acid (GABA)(B) receptor by the specific agonist baclofen blocks synaptic transmission. The action of baclofen was associated with a change in paired pulse facilitation indicating that it resulted from a reduction in the probability of transmitter release. In contrast, no change in paired pulse facilitation was observed following the induction of long-term depression. These results show that activation of GABA(B) receptors and long-term depression reduce transmitter release by distinct mechanisms.

EEG as a measure of developmental changes in the chicken brain.

Scalp-applied recording electrodes were used to monitor changes in basal EEG patterns in chickens during posthatch development. Frequency spectra produced by Fast Fourier Transform show a biphasic morphology in all chickens with one peak at about 6 Hz and another at 26 Hz. Changes in the lower frequency band show progressive development and provide a possible index of brain development. Both amplitude and dominant frequency of the spectra decrease between Weeks 3 and 8 posthatch, reaching adult levels between Weeks 5 and 7. These results suggest that modifications of basal EEG reflect the widespread functional changes in neuronal circuits occurring in chicken during the "synapse maturation" period between 3 and 8 weeks' posthatch.

Phosphorylation of proteins in chick ciliary ganglion under conditions that induce long-lasting changes in synaptic transmission: phosphoprotein targets for nitric oxide action.

Production of nitric oxide and the activation of protein kinases are required for long-term potentiation of synaptic transmission at the giant synapses in chicken ciliary ganglion. In the present study, we investigated the ability of nitric oxide to regulate the phosphorylation of endogenous proteins under conditions that induced long-term potentiation in intact ciliary ganglion and the protein kinases responsible for the phosphorylation of these proteins in lysed ciliary ganglion. Using Calcium Green-1 we showed that the nitric oxide donor sodium nitroprusside did not change the intraterminal Ca2+ dynamics in ciliary ganglion. Two dimensional phosphopeptide analysis of 32Pi-labelled intact ciliary ganglion showed that the sodium nitroprusside (300 microM) increased the phosphorylation of several phosphopeptides (P50a, P50b and P41) derived from proteins at 50,000 and 41,000 mol. wts which we have called nitric oxide-responsive phosphoproteins. A similar stimulation of phosphorylation was achieved by 8-bromo-cyclic AMP (100 microM), which also induced long-term potentiation, but not by phorbol dibutyrate (2 microM) that does not induce long-term potentiation in ciliary ganglion. When subcellular fractions from lysed ciliary ganglion were labelled in vitro by [gamma-32P]ATP in the presence of purified cGMP-dependent, cAMP-dependent or Ca2+-phospholipid-dependent protein kinases, we identified cyclic GMP-dependent protein kinase substrates that gave rise to phosphopeptides co-migrating with P50a, P50b and P41 from 32Pi-labelled intact ciliary ganglion. P50a and P41 were derived from soluble proteins while P50b was derived from a membrane-associated protein. The proteins giving rise to P50a, P50b and P41 were also substrates for cyclic AMP-dependent protein kinase, but not for calcium and phospholipid-dependent protein kinase in vitro, suggesting that nitric oxide-responsive phosphoproteins are convergence points in information processing in vivo and their phosphorylation might represent an important mechanism in nitric oxide-mediated synaptic plasticity in ciliary ganglion.

Alpha and beta subunits of CaM-kinase II are localized in different neurons in chick ciliary ganglion.

The ciliary ganglion of the chicken contains only two types of neurons. Using monoclonal antibodies against the alpha and the beta subunits of Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) we found that the alpha-subunit was localized to the choroid neurons while beta subunit was associated with the ciliary neurons. As both neurons receive their inputs from the oculomotor nerve, while their postganglionic axons leave via different nerves, the ciliary ganglion of the chicken is a neuronal system in which the functional differences between alpha and beta CaMPK-II homopolymers in the regulation of synaptic transmission can be investigated.

Developmental regulation of protein phosphatase types 1 and 2A in post-hatch chicken brain.

The activity and subcellular distribution of protein phosphatases 1 and 2A were measured in chicken forebrain and cerebellum during post-hatch development. At all post-hatch ages, a large proportion of PP1 and PP2A was membrane bound and these enzymes were less active than their cytosolic counterparts. The protein concentration of PP1 in the membranes increased 40% between 2 and 14 days and a further 60% between 14 days and adult, whereas the PP1 enzyme activity in the membranes progressively decreased. In contrast to PP1, the protein concentration of PP2A remained constant in all fractions during post-hatch development, and the enzyme activity of PP2A did not change except for a decrease in the membrane-bound activity between 2 and 14 days. These results show that the subcellular distribution and activity of PP1 is selectively regulated during post-hatch development and that membrane association and inactivation of PP1 are independent events.

Protein phosphorylation in fast and slow chicken skeletal muscles: effect of denervation.

We have identified proteins in adult chicken skeletal muscle whose phosphorylation can be used as markers for the mature fast and slow muscle phenotype. These include phosphorylase, phosphorylase kinase, and a cyclic adenosine 3',5'-monophosphate (cAMP)-stimulated, calmodulin-inhibited 28-kDa band (markers for fast muscle), a calmodulin-stimulated 50-kDa band, and two cAMP-stimulated bands at 44 and 46 kDa (markers for slow muscle), and the relative concentrations of the regulatory subunits of cAMP-dependent protein kinase (RI and RII). After denervation the pattern of phosphorylation in fast muscle changed to resemble that of slow muscle: phosphorylation of the fast phenotype markers decreased; the slow phenotype markers, barely detectable in normal fast muscle, appeared as significant phosphoproteins; and the concentration of RII increased with no change in RI. This is consistent with denervation-induced changes observed using other phenotypic markers and indicates the potential for using these phosphoprotein markers in studies of muscle development and pathophysiology.

Long-term potentiation of synaptic transmission in the avian hippocampus.

The avian hippocampus plays a pivotal role in memory required for spatial navigation and food storing. Here we have examined synaptic transmission and plasticity within the hippocampal formation of the domestic chicken using an in vitro slice preparation. With the use of sharp microelectrodes we have shown that excitatory synaptic inputs in this structure are glutamatergic and activate both NMDA- and AMPA-type receptors on the postsynaptic membrane. In response to tetanic stimulation, the EPSP displayed a robust long-term potentiation (LTP) lasting >1 hr. This LTP was unaffected by blockade of NMDA receptors or chelation of postsynaptic calcium. Application of forskolin increased the EPSP and reduced paired-pulse facilitation (PPF), indicating an increase in release probability. In contrast, LTP was not associated with a change in the PPF ratio. Induction of LTP did not occlude the effects of forskolin. Thus, in contrast to NMDA receptor-independent LTP in the mammalian brain, LTP in the chicken hippocampus is not attributable to a change in the probability of transmitter release and does not require activation of adenylyl cyclase. These findings indicate that a novel form of synaptic plasticity might underlie learning in the avian hippocampus.

Presynaptic long-term depression at a central glutamatergic synapse: a role for CaMKII.

CaMKII is a calcium-activated kinase that is abundant in neurons and has been strongly implicated in memory and learning. Here we show that low-frequency stimulation of glutamatergic afferents in hippocampal slices from juvenile domestic chicks results in long-term depression of synaptic transmission. This reduction does not require activation of NMDA or metabotropic glutamate receptors and does not require a rise in postsynaptic calcium. However, buffering presynaptic calcium prevents the reduction of the excitatory postsynaptic potential or current that is induced by low-frequency stimulation. In addition, application of the calmodulin antagonist calmidazolium, or the specific CaMKII antagonist KN-93, completely blocks long-term depression. These findings demonstrate a newly discovered form of long-term synaptic depression in the avian hippocampus.

In vitro phosphorylation of medial vestibular nucleus and prepositus hypoglossi proteins during behavioural recovery from unilateral vestibular deafferentation in the guinea pig.

Unilateral removal of vestibular nerve input to the vestibular nuclei (e.g. by unilateral labyrinthectomy, UL) results in severe ocular motor and postural disorders which disappear over time (vestibular compensation). We investigated whether recovery of ocular motor function is temporally correlated with changes in protein phosphorylation in the medial vestibular nucleus (MVN) and prepositus hypoglossi (PH; MVN/PH) in vitro. Bilateral MVN/PH were dissected from 48 guinea pigs following decapitation at 10 h, 53 h or 2 weeks post-UL, or -sham operation and frozen. Tissue extracts were incubated with [gamma-32P]ATP +/- Ca2+ plus phorbol 12,13-dibutyrate and phosphatidylserine. UL resulted in a significant bilateral increase in the 32P-incorporation into a 65-85 kDa band (probably the myristoylated alanine-rich C kinase substrate, MARCKS) in compensated animals (53 h post-UL) under conditions which favoured the activation of protein kinase C. Under identical conditions, the labelling of a 42-49 kDa protein (P46) was increased significantly in the bilateral MVN/PH between either 10 h or 53 h and 2 weeks post-UL; there were no significant changes over time in sham controls. These results show that later stages of vestibular compensation are accompanied by changes in the phosphorylation of several likely protein kinase C substrates in the MVN/PH in vitro.

Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.

Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.

Effect of hypothyroidism on the subcellular distribution of Ca2+/calmodulin-stimulated protein kinase II in chicken brain during posthatch development.

In developing chicken brain Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T3) at 42 days. The concentrations of alpha- and beta-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca2+/calmodulin- or ZN2+-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca2+/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

Developmental changes in glutamate receptor stimulated inositol phospholipid metabolism and 45Ca(2+)-accumulation in posthatch chicken forebrain.

In chicken forebrain the two phases of synapse development, formation and maturation, are temporally well separated. We have used this model system to determine the developmental profile of glutaminergic activation of phosphoinositidase C. Stimulation of [3H]inositol-loaded forebrain prisms by quisqualic acid (QA; 30 microM), or the metabotropic agonist 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD; 30 microM), significantly increased [3H]inositol phosphate production. This response progressively decreased with developmental age, with the largest (approximately 3-fold) decrease occurring between 21 days and adult (> 10 weeks). In contrast, QA (30 microM) stimulated a quite distinct developmental profile for 45Ca2+ accumulation, with the response being maximal between 7 and 14 days before declining sharply to adult levels by 21-25 days. These results demonstrate that there is a major decrease in metabotropic glutamate receptor activation of phosphoinositidase C during the maturation phase of synapse development.