Michael L. Berger

Induction of stress protein HSP70 in nerve cells after status epilepticus in the rat

The accumulation of the stress protein HSP70 was found to be an excellent marker for prolonged seizure related metabolic activity of neurons. After kainic acid (KA) induced status epilepticus we observed HSP70 immunoreactivity in the hippocampal CA4 and CA1 sectors, the subiculum, the basolateral and the lateral nuclei of the amygdala, the mediodorsal nucleus of the thalamus, the caudal part of the striatum, the claustrum and in neurons of certain neocortical areas. HSP70-positive nerve cells appeared normal in conventional histological stains. Conversely, degenerating neurons (e.g. in the hippocampal CA3 sector) remained unlabeled.

Regional heterogeneity in the distribution of neurotransmitter markers in the rat hippocampus.

A detailed neurochemical analysis of the distribution of markers for the most relevant neurotransmitter systems within the rat hippocampal formation has been performed. The hippocampi, obtained from unfrozen brains of male Sprague-Dawley rats were subdissected into tissue parts containing mainly CA1, CA3 or the dentate gyrus, respectively. Each part was further divided into ventral and dorsal halves. In these six hippocampal subregions the concentrations of noradrenaline, dopamine, serotonin, 3-methoxy-4-hydroxyphenylglycol, 5-hydroxyindoleacetic acid and the putative neurotransmitter amino acids glutamate, aspartate, GABA, glycine and taurine, and the levels of somatostatin and neuropeptide Y and the activities of choline acetyltransferase, acetylcholinesterase and glutamate decarboxylase were measured. A marked heterogeneity in the subregional distribution of markers for various neurotransmitter systems within the hippocampal formation was observed. Each neuronal marker was characterized by an individual pattern of distribution. Most of the markers showed a concentration-gradient, increasing from dorsal to ventral; only taurine was more abundant in the dorsal than in the ventral parts and no dorsoventral difference was seen for aspartate, glycine and neuropeptide Y. The highest molar ratios of total 3-methoxy-4-hydroxyphenylglycol to noradrenaline and 5-hydroxyindoleacetic acid to serotonin were found in the dorsal hippocampus. The levels of noradrenaline, GABA and glutamate decarboxylase activity were highest in the dentate gyrus and lowest in CA1. The concentrations of somatostatin were highest in CA1; those of serotonin were highest in CA3. Highest activities of choline acetyltransferase and acetylcholinesterase were found in the dentate gyrus; lowest activities were found in CA3. In CA3 the lowest values of glutamate, aspartate, taurine and somatostatin were also found. The heterogeneity in the distribution of individual neurochemical markers allows insights into possible functional differences of hippocampal subregions and provides a relevant basis for future neurochemical investigations in this brain area.

Kainic acid-induced changes of serotonin and dopamine metabolism in the striatum and substantia nigra of the rat.

Levels of the putative neurotransmitters serotonin (5-HT) and dopamine (DA) and their respective metabolites 5-hydroxyindoleacetic acid (5-HIAA) and dihydroxyphenylacetic acid (DOPAC) was determined in the rat striatum after unilateral intrastriatal injection of the convulsive neurotoxin kainic acid. Two days after intrastriatal kainic acid injection, levels of the 5-HT metabolite 5-HIAA were increased by abut 200% in the injected striatum and by about 150% in the contralateral striatum. An elevated striatal 5-HIAA content was still detectable 10 days after the kainate lesion, but approached normal values 10 weeks after the injection of the neurotoxin. Two days after the lesion, but not at the other time intervals, a moderate increase of 5-HIAA also occurred bilaterally in other brain areas such as the substantia nigra, frontal cortex and hypothalamus. Levels of 5-HT were decreased significantly in the injected striatum 2 days after the intrastriatal application of kainic acid and increased by about 40% after 10 weeks. The 5-HT concentration in the contralateral striatum or in the three other brain areas examined was unchanged at all time intervals. Levels of the DA metabolite DOPAC and DA turnover were increased in the lesioned striatum 2 days after kainic acid injection; concomitantly the DOPAC level was increased in the substantia nigra of the contralateral side. DOPAC levels of the contralateral striatum were unchanged or slightly reduced 2 days after the injection. Ten days as well as 10 weeks after the lesion there was a slightly increased DOPAC concentration in both striata. The levels of DA were not altered at any time interval after the injection of kainic acid.

An estimate of the impact of Stratosphere to Troposphere Transport (STT) on the lower free tropospheric ozone over the Alps using 10Be and 7Be measurements

Jungfraujoch (JUN), Switzerland and Zugspitze (ZUG), Germany. Inspection of the variability of the ratio 10 Be/ 7 Be in relation to 10 Be, 7 Be, and relative humidity (RH) reveals that the ratio is independent from processes that have a clear effect on both radionuclides, such as wet scavenging. High ratio values are generally met under cyclonic or northerly advective conditions, which are the synoptic situations mostly related to stratosphere-to-troposphere transport (STT) events over central Europe, while the 10-day back trajectories indicate a stratospheric source for the majority of the cases within the upper 10% quantile of 10 Be/ 7 Be ratios. The monthly 10 Be/ 7 Be ratios show a clear May and June peak at JUN and a much weaker seasonality at ZUG. A simple mixing model is used for an independent estimate of the strength of STT throughout the year based on the 7 Be and 10 Be measurements. In spite of the various uncertainties, the results indicate a seasonal cycle of stratospheric ozone percentage contribution with an early spring maximum (3– 11%) and autumn minimum (1–2%) at ZUG, while at JUN, a primary maximum in May and June (6–18%), a secondary maximum in March (4–13%), and a minimum again in autumn (1–4%) are revealed. Although the simple method applied here provides an independent estimate for the impact of STT to the lower troposphere, it nevertheless shows relatively good agreement with Lagrangian model calculations, especially for ZUG. INDEX TERMS: 0341 Atmospheric Composition and Structure: Middle atmosphere—constituent transport and chemistry (3334); 0365 Atmospheric Composition and Structure: Troposphere—composition and chemistry; 0368 Atmospheric Composition and Structure: Troposphere—constituent transport and chemistry; 3329 Meteorology and Atmospheric Dynamics: Mesoscale meteorology; 3362 Meteorology and Atmospheric Dynamics: Stratosphere/troposphere interactions; KEYWORDS: stratospheric intrusions, cosmogenic radionuclides, tropospheric ozone, 7 Be, 10 Be, Alps Citation: Zanis, P., et al., An estimate of the impact of stratosphere-to-troposphere transport (STT) on the lower free tropospheric ozone over the Alps using 10 Be and 7 Be measurements, J. Geophys. Res., 108(D12), 8520, doi:10.1029/2002JD002604, 2003.

Changes in the brain protein levels following administration of kainic acid.

Kainic acid (KA), a potent neurotoxin and excitatory amino acid, leads to derangements and modulation of brain proteins. No global brain protein expression pattern induced by KA‐treatment has been reported yet. We therefore studied the effect of systemic KA administration on the levels of brain proteins. Rats were injected placebo or KA intraperitoneally and brain was taken after one week. The mitochondrial and cytosolic fractions of the brain proteins were analyzed by proteomics technologies and the levels of selected proteins were quantified using specific software. Heat shock protein HSP 27 was exclusively detected in brains of animals treated with KA, whereas the glucose regulated protein GRP 78 was downregulated. The levels of neurofilaments and alpha‐internexin were significantly decreased and a fragment of tubulin alpha‐1 chain was manifold increased in KA‐brains. The mitochondrial enzymes dihydrolipoamide dehydrogenase, ATP synthase beta chain and isocitrate dehydrogenase were reduced and pyruvate kinase M1 was increased following KA treatment. We conclude that the concomitant determination of the brain proteins indicates altered regulation of heat shock proteins, neuronal death, cytoskeletal disruption, and mitochondrial derangement by systemic KA administration. This report confirms and extends previous studies on the effect of KA on the expression of brain proteins and suggests that our analytical system can serve as a model for neurotoxicological, neurobiological, and neuropathological proteome studies.

Effects of NMDA receptor modulators on a blood-brain barrier in vitro model

Changes of the functionality of the blood-brain barrier (BBB) have been reported in the context of several brain related diseases such as multiple sclerosis, epilepsy, Alzheimers disease and stroke. Several publications indicated the presence and functionality of the NMDA receptor (NMDAR) at the brain endothelium and a possible involvement of the NMDAR in the above-mentioned diseases. Recently, it was shown that the application of the NMDAR antagonist MK801 can block several adverse effects at the BBB in vitro, but also that MK801 can significantly change the proteome of brain endothelial cells without simultaneous stimulation of NMDAR by glutamate. Based on these reports we investigated if NMDAR antagonists MK801 and D-APV can affect the intracellular calcium level (Ca²⁺i) of an in vitro BBB model based on human cell line ECV304 on their own and compared these results to effects mediated by NMDAR agonists glutamate and NMDA. Treatment of ECV304 cells for 30 min with glutamate resulted in no significant change of Ca²⁺i. On the contrary, application of NMDA and NMDAR antagonists D-APV and MK801 led to a significant and concentration dependent decrease of Ca²⁺i. Further studies revealed that glutamate was able to decrease the transendothelial electrical resistance (TEER) of the BBB in vitro model, whereas NMDA and D-APV were able to increase TEER. Analysis of the protein expression levels of tight junctional molecules ZO-1 and occludin showed a complex regulation after application of NMDAR modulators. In summary, it was shown that NMDAR antagonists can alter BBB key properties in vitro on their own. Moreover, although qPCR results confirmed the presence of NMDA receptor subunits NR1, NR2A, NR2B and NR2C, membrane binding studies failed to prove the typical plasma membrane localization and functionality in human BBB cell line ECV304.

Chronic exposure to manganese decreases striatal dopamine turnover in human alpha-synuclein transgenic mice

Interaction of genetic and environmental factors is likely involved in Parkinsons disease (PD). Mutations and multiplications of alpha-synuclein (α-syn) cause familial PD, and chronic manganese (Mn) exposure can produce an encephalopathy with signs of parkinsonism. We exposed male transgenic C57BL/6J mice expressing human α-syn or the A53T/A30P doubly mutated human α-syn under the tyrosine hydroxylase promoter and non-transgenic littermates to MnCl₂-enriched (1%) or control food, starting at the age of 4 months. Locomotor activity was increased by Mn without significant effect of the transgenes. Mice were sacrificed at the age of 7 or 20 months. Striatal Mn was significantly increased about three-fold in those exposed to MnCl₂. The number of tyrosine hydroxylase positive substantia nigra compacta neurons was significantly reduced in 20 months old mice (-10%), but Mn or transgenes were ineffective (three-way ANOVA with the factors gene, Mn and age). In 7 months old mice, striatal homovanillic acid (HVA)/dopamine (DA) ratios and aspartate levels were significantly increased in control mice with human α-syn as compared to non-transgenic controls (+17 and +11%, respectively); after Mn exposure both parameters were significantly reduced (-16 and -13%, respectively) in human α-syn mice, but unchanged in non-transgenic animals and mice with mutated α-syn (two-way ANOVA with factors gene and Mn). None of the parameters were changed in the 20 months old mice. Single HVA/DA ratios and single aspartate levels significantly correlated across all treatment groups suggesting a causal relationship between the rate of striatal DA metabolism and aspartate release. In conclusion, under our experimental conditions, Mn and human α-syn, wild-type and doubly mutated, did not interact to induce PD-like neurodegenerative changes. However, Mn significantly and selectively interacted with human wild-type α-syn on indices of striatal DA neurotransmission, the neurotransmitter most relevant to PD.

Zinc regulates the dopamine transporter in a membrane potential and chloride dependent manner

The dopamine transporter (DAT), a membrane protein specifically expressed by dopaminergic neurons and mediating the action of psychostimulants and dopaminergic neurotoxins, is regulated by Zn(2+) which directly interacts with the protein. Herein, we report a host-cell-specific direction of the Zn(2+) effect on wild type DAT. Whereas low mumolar Zn(2+) decreased dopamine uptake by DAT expressing HEK293 cells, it stimulated uptake by DAT expressing SK-N-MC cells. Inhibition or stimulation was lost in a DAT construct without the binding site for Zn(2+). Also reverse transport was differentially affected by Zn(2+), dependent on whether the DAT was expressed in HEK293 or SK-N-MC cells. Pre-treatment of DAT expressing cells with phorbol-12-myristate-13-acetate, an activator of protein kinase C, attenuated the inhibitory effect of Zn(2+) on uptake in HEK293 cells and increased the stimulatory effect in SK-N-MC cells. Patch-clamp experiments under non-voltage-clamped conditions revealed a significantly higher membrane potential of HEK293 than SK-N-MC cells and a reduced membrane potential after phorbol ester treatment. Lowering chloride in the uptake buffer switched the stimulatory effect of Zn(2+) in SK-N-MC cells to an inhibitory, whereas high potassium depolarization of HEK293 cells switched the inhibitory effect of Zn(2+) to a stimulatory one. This study represents the first evidence that DAT regulation by Zn(2+) is profoundly modulated by the membrane potential and chloride.

Autoradiography with [3H]PK11195 of spinal tract degeneration in amyotrophic lateral sclerosis

Abstract. The diagnostic hallmarks of amyotrophic lateral sclerosis (ALS) are degeneration of upper and lower motor neurons and of corticospinal tracts. Here, we demonstrate the suitability of the gliosis marker [3H]PK11195 for quantitative evaluation of tract degeneration in ALS in vitro. Binding of [3H]PK11195 was increased in lateral and ventral white matter of ALS spinal cords but not in the anterior horn, in spite of a dramatic loss in muscarinic binding sites and a high level of oxidatively modified protein. Labeling of activated microglia with [11C]PK11195 may also allow tract degeneration in ALS to be visualized in vivo.

NMDA receptor affinities of 1,2-diphenylethylamine and 1-(1,2-diphenylethyl)piperidine enantiomers and of related compounds.

We resolved 1,2-diphenylethylamine (DPEA) into its (S)- and (R)-enantiomer and used them as precursors for synthesis of (S)- and (R)-1-(1,2-diphenylethyl)piperidine, flexible homeomorphs of the NMDA channel blocker MK-801. We also describe the synthesis of the dicyclohexyl analogues of DPEA. These and related compounds were tested as inhibitors of [(3)H]MK-801 binding to rat brain membranes. Stereospecificity ranged between factors of 0.5 and 50. Some blockers exhibited stereospecific sensitivity to the modulator spermine. Our results may help to elucidate in more detail the NMDA channel pharmacophore.