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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.23, #1-3)

Amino acids in neurobiology: Neuroprotective and neurotoxic aspects of amino acids involved in neurotransmission and neuromodulation – General Introduction by B. D. Kretschmer; W. J. Schmidt; R. M. Kostrzewa; M. Herrera-Marschitz (pp. 1-7).

Excitotoxicity – Introduction by A. Novelli; R. A. Tasker (pp. 9-10).

Co-activation of the phosphatidylinositol-3-kinase/Akt signaling pathway by N-methyl-D-aspartate and TrkB receptors in cerebellar granule cell neurons by D. Zhu; R. H. Lipsky; A. M. Marini (pp. 11-17).

Neuroprotective concentrations of N-methyl-D-aspartate (NMDA) promote survival of cerebellar granule cell neurons against glutamate excitotoxicity through a TrkB receptor-mediated brain-derived neurotrophic factor (BDNF) autocrine loop. However, the intracellular signaling pathway(s) are not clear. Our results show that PI-3 kinase/Akt is activated by either NMDA or BDNF displaying differential kinetics. BDNF and NMDA increased Akt phosphorylation within 5 minutes but maximal activation by NMDA was observed at 3 hours. Akt phosphorylation was completely blocked by the PI-3 kinase inhibitor LY294002. NMDA-mediated activation of Akt was completely blocked by MK-801 and partially blocked by the TrkB receptor inhibitor, K252a, indicating the requirement of TrkB receptors for maximal activation by NMDA. In contrast, BDNF-induced Akt phosphorylation was abolished by K252a, but not by the addition of MK-801. Therefore, the PI-3 kinase/Akt pathway is co-activated by NMDA and TrkB receptors. The kinetics of BDNF and NMDA-mediated activation of PI-3 kinase/Akt suggests that they have different roles in intraneuronal time-related events.

Keywords: Keywords: N-methyl-D-aspartate; Brain-derived neurotrophic factor; Rat cerebellar granule cells; Phosphatidylinositol 3-kinase/Akt; TrkB receptor; Neuroprotection

Inhibition of phosphatidylcholine synthesis is associated with excitotoxic cell death in cerebellar granule cell cultures by T. Gasull; N. DeGregorio-Rocasolano; M. Enguita; J. M. Hurtan; R. Trullas (pp. 19-25).

Glucose deprivation (GD) enhances the sensitivity of cerebellar granule cells to die by excitotoxicity. Neither 70 min of GD, a treatment that depletes cell energy resources, nor exposure to 20 μM glutamate (GLU) for 30 min, induce significant cell death in cultures of cerebellar granule cells. However, the combined treatment with GLU and GD induces choline (Cho) release before excitotoxic cell death. We investigated whether the neurotoxic effect of this treatment is related with inhibition of phosphatidylcholine (PC) synthesis. We found that exposure to GLU for 30 min, to GD for 70 min, and to the combination of both, inhibited PC synthesis at the end of treament by 71%, 92% and 91%, respectively. The inhibition of PC synthesis was accompanied by a decrease in the incorporation of [³H]Cho into phosphocholine and by an increase of the intracellular content of free [³H]Cho, indicating that these treatments inhibit the synthesis of PC by inhibiting choline kinase activity. However, only the combined treatment with GLU and GD induced a prolonged inhibition of PC synthesis that extented after the end of treatment. These results show that excitotoxic death is associated with sustained inhibition of PC synthesis and suggest that this effect of the combined treatment with GLU and GD on PC synthesis is produced by an action on an enzymatic step downstream of choline kinase activity.

Keywords: Keywords: Excitotoxicity; Neuronal death; Phosphatidylcholine; Glutamate

Expression of cell-cycle-related proteins and excitoxicity by D. Uberti; E. Meli; M. Memo (pp. 27-30).

Previous work from our laboratory has suggested the functional contribution of p53 to the cascade of events triggered by excitatory amino acids and leading to cell death in primary neurons. Here we show that this paradigm can be extended to cortical neurons treated with NMDA. We found that exposure of the cells to either 300 μM or 2 mM NMDA induced an enhancement of p53 protein levels which was already significant at 60 min after the lesion, while very low staining of the protein was observed in untreated cells. The effect was time- and concentration-dependent, reaching the maximal induction at 3 h. NMDA treatment also resulted in an increase of gadd45 protein levels which was evident in both treatment at 3 h, the time when p53 was maximally induced.Our data give further evidence suggesting that a repertoire of events typical of proliferating cells is activated in degenerating neurons.

Keywords: Keywords: Cortical neurons; Cyclin-dependent kinase; Excitotoxicity; Glutamate; Neurodegenerative diseases; Tumour suppressor gene; Gadd45

Nefopam, an analogue of orphenadrine, protects against both NMDA receptor-dependent and independent veratridine-induced neurotoxicity by M. T. Fernández-Sánchez; R. Díaz-Trelles; A. Groppetti; B. Manfredi; A. T. Brini; G. Biella; M. L. Sotgiu; A. Novelli (pp. 31-36).

Nefopam hyghochloride is a potent analgesic compound commercialized in most Western Europe for 20 years, which possesses a profile distinct from that of opioids or anti-inflammatory drugs. Previous evidence suggested a central action of nefopam but the detailed mechanisms remain unclear. While, nefopam structure resembles that of orphenadrine, an uncompetitive NMDA receptor antagonist, here we report that differently from orphenadrine, nefopam (100 μM) failed to protect cultured cerebellar neurons from excitotoxicity following direct exposure of neurons to glutamate. Moreover, nefopam failed to displace MK-801 binding to hippocampal membranes. Nefopam effectively prevented NMDA receptor-mediated early appearance (30 min) of toxicity signs induced by the voltage sensitive sodium channel (VSSC) activator veratridine. The later phase (24 h) of neurotoxicity by veratridine occurring independently from NMDA receptor activation, was also prevented by nefopam. Nefopam effect was not mimicked by the GABA receptor agonist muscimol.

Keywords: Keywords: Excitotoxicity; NMDA receptor antagonist; Voltage sensitive sodium channels; Cerebellar neurons in culture

Domoic acid neurotoxicity in hippocampal slice cultures by B. Jakobsen; A. Tasker; J. Zimmer (pp. 37-44).

The neurotoxicity of domoic acid was studied in 2–3 week old rat hippocampal slice cultures, derived from 7 day old rat pups. Domoic acid 0.1–100 μM was added to the culture medium for 48 hrs, alone or together with the glutamate receptor antagonists NS-102 (5-Nitro-6,7,8,9-tetrahydrobenzo[G]indole-2,3-dione-3-oxime), NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) or MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine hydrogen maleate), followed by transfer of the cultures to normal medium for additional 48 hrs. Neuronal degeneration in the fascia dentata (FD), CA3 and CA1 hippocampal subfields was monitored and EC₅₀ values estimated by densitometric measurements of the cellular uptake of propidium iodide (PI). The CA1 region was most sensitive to domoic acid, with an EC₅₀ value of 6 μM domoic acid, estimated from the PI-uptake at 72 hrs. Protective effects of 10 μM NBQX against 3 and 10 μM domoic acid were observed for both dentate granule cells and CA1 and CA3c pyramidal cells. NS102 and MK 801 only displayed protective effects when combined with NBQX. MK801 significantly increased the combined neuroprotective effect of NBQX and NS102 against 10 μM domoic acid in both CA1 and FD, but not in CA3. We conclude, that domoic acid neurotoxicity in CA3 and in hippocampal slice cultures in general primarily involves AMPA/kainate receptors. At high concentrations (10 μM domic acid) NMDA receptors are, however, also involved in the toxicity in CA1 and FD.

Keywords: Keywords: Domoic acid; NBQX; NS102; Propidium iodide; Hippocampal slice cultures

Comparison of the in vitro and in vivo neurotoxicity of three new sources of kainic acid by R. A. R. Tasker; P. B. Bernard; T. A. Doucette; D. S. Kerr; Y. Zabidin; L. Alvarez-Fernandez; B. Fernandez-Maroto; M. T. Fernandez-Sanchez; A. Novelli (pp. 45-54).

Historically, all commercially available kainic acid has been derived from a single biological source using a consistent method of extraction and purification. That source became unavailable in 1995. Recently, three new commercial suppliers of kainic acid have made the product available, but the source of the material and the purification processes used differ. Our objective was to systematically compare the response produced by each of these new sources of kainic acid using three established neurobiological techniques: neuronal cell culture, hippocampal slice electrophysiology, and whole animal behavioural toxicity.Results in all three systems indicated no overall differences between the three formulations, although studies in both cerebellar neuron cultures and whole animal toxicity testing in mice, revealed some significant differences that may imply subtle differences in receptor selectivity and/or potency.We conclude that all three sources of kainic acid are viable alternatives to traditional kainate but they may not be identical. Until further information becomes available researchers may want to avoid using the three formulations interchangeably, and take note of the source of kainic acid when evaluating literature describing results from other laboratories.

Keywords: Keywords: Glutamate; Excitatory amino acids; non-NMDA receptors; Excitotoxicity

Novel roles of amino acids and analogues in neurodegenerative disorders – Introduction by R. M. Kostrzewa; J. P. Kostrzewa; R. Brus (pp. 55-56).

Neuroprotective and neurotoxic roles of levodopa (L-DOPA) in neurodegenerative disorders relating to Parkinson's disease by R. M. Kostrzewa; J. P. Kostrzewa; R. Brus (pp. 57-63).

Despite its being the most efficacious drug for symptom reversal in Parkinson's disease (PD), there is concern that chronic levodopa (L-DOPA) treatment may be detrimental. In this paper we review the potential for L-DOPA to 1) autoxidize from a catechol to a quinone, and 2) generate other reactive oxygen species (ROS). Overt toxicity and neuroprotective effects of L-DOPA, both in vivo and in vitro, are described in the context of whether L-DOPA may accelerate or delay progression of human Parkinson's disease.

Keywords: Keywords: L-DOPA; Parkinson's disease; Reactive oxygen species

L-DOPA: From a biologically inactive amino acid to a successful therapeutic agent by O. Hornykiewicz (pp. 65-70).

The article traces the development of research on the naturally occurring amino acid L-3,4-dihydroxyphenylalanine (L-dopa), from the first synthesis of its D,L racemate in 1911, and the isolation of its L-isomer from seedling of Vicia faba beans to the amino acid's successful application, from 1961 onward, as the most efficacious drug treatment of Parkinson's disease (PD). Upon its isolation from legumes in 1913, L-dopa was declared to be biologically inactive. However, two early pharmacological studies, published in 1927 and 1930 respectively, proved (in the rabbit) that D,L-dopa exerted significant effects on glucose metabolism (causing marked hyperglycemia) and on arterial blood pressure. Interest in L-dopa's biological activity increased considerably following the discovery, in 1938, of the enzyme L-dopa decarboxylase and the demonstration that in the animal and human body L-dopa was enzymatically converted to dopamine (DA), the first biologically active amine in the biosynthetic chain of tissue catecholamines. This prompted, in the 1940s, many studies, both in animals and in humans, especially concerned with the vasopressor potential of L-dopa/DA. In the 1950s, the focus of L-dopa research shifted to its potential for replenishing the experimentally depleted (by insulin or reserpine) peripheral and brain catecholamine stores and the concomitant restoration of normal function. During that period, of special interest were the observations that L-dopa reversed the reserpine-induced state of “tranquilisation” and that its decarboxylation product DA occurred in high amounts in animal and human brain, with a preferential localization in the basal ganglia. These observations set the stage for the beginning of DA studies in PD brain. In 1960, the severe brain DA deficit, confined to patients with PD was discovered, and a year later L-dopa's strong therapeutic effect in patients with PD was demonstrated. In 1967, the chronic high-dose oral L-dopa regimen was successfully introduced into clinical practice. Despite some initial doubts about L-dopa's mechanism of action in PD, it is now generally recognized that L-dopa use in PD is a classic example of a brain neurotransmitter replacement therapy. However, the DA replacement potential of L-dopa may not be its sole action of interest, as suggested by recent evidence that L-dopa may also have its own biological activity in the CNS, independent of DA.

Keywords: Keywords: L-Dopa history; L-Dopa pharmacology; Dopamine; Dopamine replacement; L-Dopa therapy; Parkinson's disease

Restorative effects of glutamate antagonists in experimental parkinsonism by T. Archer; T. Palomo; A. Fredriksson (pp. 71-85).

Several compounds with antagonistic actions on N-methyl-D-aspartate (NMDA) receptors were tested for an antiakinesic action in hypoactive MPTP-treated C57 BL/6 mice rendered tolerant to the motor activity enhancing effects of the 20 mg/kg, s.c., dose of L-Dopa; each compound was administered 60 min before the administration of the dopamine precursor. The classes of compounds studied included the noncompetitive NMDA antagonists, memantine, amantadine and MK-801, the competitive NMDA antagonist, CGP 40116, the anticonvulsive and putative anticonvulsive agents, lamotrigine and FCE 26743, with a partial glutamatergic antagonistic action. All six compounds elevated locomotor, rearing and total activity counts of L-Dopa-tolerant mice in co-administration with L-Dopa in dose-specific or dose-dependent manners but only memantine and MK-801 affected motor activity in the control mice, that also received chronic L-Dopa treatment. Thus, the restorative actions of those compounds in suprathreshold L-Dopa-tolerant MPTP-treated mice subjected to “wearing-off” of L-Dopa efficacy were assessed in a series of experiments. Within each class of potentially therapeutic agents a differential restorative efficacy of the motor activity-stimulating effects of hypoactive MPTP mice was obtained, confirming the putative antiparkinsonian applications of compounds with glutamate antagonistic actions.

Keywords: Keywords: MPTP; Hypokinesia; Locomotion; Rearing; Memantine; Amantadine; MK-801; CGP 40116; Lamotrigine; FCE 26743; Chronic administration; L-Dopa; 20 mg/mg; Co-administration; Hyperactivity; Motor fluctuations; “Wearing-off”; Drug-tolerance; Synergism; Restoration; C57 BL/6 mice

Modeling Alzheimer's disease and other proteopathies in vivo: Is seeding the key? by L. C. Walker; F. Bian; M. J. Callahan; W. J. Lipinski; R. A. Durham; H. LeVine (pp. 87-93).

Protein misfolding and aberrant polymerization are salient features of virtually all central neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease, triplet repeat disorders, tauopathies, and prion diseases. In many instances, a single amino acid change can predispose to disease by increasing the production and/or changing the biophysical properties of a specific protein. Possible pathogenic similarities among the cerebral proteopathies suggest that therapeutic agents interfering with the proteopathic cascade might be effective against a wide range of diseases. However, testing compounds preclinically will require disease-relevant animal models. Numerous transgenic mouse models of β-amyloidosis, tauopathy, and other aspects of AD have now been produced, but none of the existing models fully recapitulates the pathology of AD. In an attempt to more faithfully replicate the human disease, we infused dilute AD-brain extracts into Tg2576 mice at 3-months of age (i.e. 5–6 months prior to the usual onset of β-amyloid deposition). We found that intracerebral infusion of AD brain extracts results in: 1) Premature deposition of β-amyloid in eight month-old, β-amyloid precursor protein (βAPP)-transgenic mice (Kane et al., 2000); 2) augmented amyloid load in the injected hemisphere of 15 month-old transgenic mice; 3) evidence for the spread of pathology to other brain areas, possibly by neuronal transport mechanisms; and 4) tau hyperphosphorylation (but not neurofibrillary pathology) in axons passing through the injection site. The seeding of β-amyloid in vivo by AD brain extracts suggests pathogenic similarities between β-amyloidoses such as AD and other cerebral proteopathies such as the prionoses, and could provide a new model for studying the proteopathic cascade and its neuronal consequences in neurodegenerative diseases.

Keywords: Keywords: Alzheimer's disease; Amyloid; Conformational disease; Huntington's disease; Parkinson's disease; Proteopathy; Senile plaque; Tau; Transgenic

Submolecular adventures of brain tyrosine: what are we searching for now? by A. Kochman; Cz. Kośka; D. Metodiewa (pp. 95-101).

This overview summarizes recent findings on the role of tyrosyl radical (TyrO^•) in the multitudinous neurochemical systems of brain, and theorizes on the putative role of TyrO^• in neurological disorders [Parkinson's disease (PD), Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS)]. TyrO^• and tyrosine per se can interact with reactive oxygen species (ROS) and reactive nitrogen species (RNS) via radical mechanisms and chain propagating reactions. The concentration of TyrO^•, ROS and RNS can increase dramatically under conditions of generalized stress: oxidative, nitrative or reductive as well, and this can induce damage directly (by lipid peroxidation) or indirectly (by proteins oxidation and/or nitration), potentially causing apoptotic neuronal cell death or autoschizis.Evidence of lesion-induced neuronal oxidative stress includes the presence of protein peroxides (TyrOOH), DT (o,o'-dityrosine) and 3-NT (3-nitrotyrosine). Mechanistic details of protein- and enzymatic oxidation/nitration in vivo remain unresolved, although recent in vitro data strongly implicate free radical pathways via TyrO^•. Nitration/denitration processes can be pathological, but they also may play: 1) a signal transduction role, because nitration of tyrosine residues through TyrO^• formation can modulate, as well the phosphorylation (tyrosine kinases activity) and/or tyrosine hydroxylation (tyrosine hydroxylase inactivation), leading to consequent dopamine synthesis failure and increased degradation of target proteins, respectively; 2) a role of “blocker” for radical-radical reactions (scavenging of NO^•, NO^• ₂ and CO₃ ^•− by TyrO^•); 3) a role of limiting factors for peroxynitrite formation, by lowering O₂ ^•− formation, which is strongly linked to the pathogenesis of neural diseases.It is still not known if tyrosine oxidation/nitration via TyrO^• formation is 1) a footprint of generalized stress and neuronal disorders, or 2) an important part of O₂ ^•− and NO^• metabolism, or 3) merely a part of integral processes for maintaining of neuronal homeostasis. The full answer to these questions should be of top research priority, as the problem of increased free radical formation in brain and/or imbalance of the ratios ROS/RNS/TyrO^• may be all important in defining whether oxidative stress is the critical determinant of tissue and neural cell injury that leads to pathological end-points.

Keywords: Keywords: Tyrosine; Oxidation; Nitration; Mechanisms; Neural disorders; Generalized stress

Mechanisms and treatment of L-DOPA-induced dyskinesias – Introduction by W. Danysz (pp. 103-103).

Transcription factors involved in the pathogenesis of L-DOPA-induced dyskinesia in a rat model of Parkinson's disease by M. A. Cenci (pp. 105-109).

L-DOPA-induced dyskinesia (abnormal involuntary movements) is one of the most debilitating complications of chron-ic L-DOPA pharmacotherapy in Parkinson's disease. It is generally agreed that dyskinesia arises as a consequence of pulsatile dopamine-receptor stimulation in the brain, causing downstream changes in genes and proteins. Advance in our understanding of such changes is critically dependent on the availability of suitable animal models. We have introduced a new method to classify and rate L-DOPA-induced abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA) lesioned rats. This method allows us to dissect the molecular correlates of a dyskinetic motor response to L-DOPA in this species. One of the most prominent molecular changes underlying the development of dyskinesia in the rat consists in the striatal induction of prodynorphin gene expression by L-DOPA. This effect is mediated by FosB-related transcription factors of 32–37 kDa, which are co-induced with prodynophin in striatal neurons of the “direct pathway”. Both AIM development and the associated upregulation of prodynorphin mRNA by L-DOPA are significantly inhibited by the intrastriatal infusion of fosB antisense. Antisense-mediated knockdown of CREB (cyclic AMP response-element binding proteins) has however no effect. Our results identify fosB as a potential target for adjunctive antiparkinsonian therapies.

Keywords: Kewords: Immediate-early genes; Movement disorder; Basal ganglia; Dynorphin; Striatonigral

Functional alteration by NMDA antagonist: Effects of L-Dopa, neuroleptics drug and postnatal administration by A. Fredriksson; T. Archer (pp. 111-132).

Antiakinsic effects of the uncompetitive NMDA antagonists, memantine, amantadine and MK-801, and competitive antagonists, CGP 40116, alone or in co-administration with acute subthreshold dose of L-Dopa (5 mg/kg) in MPTP-treated mice, functional alterations induced by acute MK-801 in combinations with neuroleptic compounds or behavioural deficits following postnatal administration of MK-801 were investigated. Memantine and amantadine injected 60 min before the subthreshold dose of L-Dopa (5 mg/kg), induced antiakinesic actions in hypokinesic MPTP-treated mice. Concurrently, higher doses of memantine and MK-801 caused dyskinesic changes, reducing further rearing (10 and 30 mg/kg) and locomotor (30 mg/kg) behaviour of the MPTP mice; MK-801 elevated locomotion (0.1 mg/kg) but reduced rearing (0.3 mg/kg). In control, saline-treated mice, memantine (3, 10 and 30 mg/kg) and MK-801 (0.1 and 0.3 mg/kg) increased locomotor behaviour but decreased rearing behaviour. In rats, MK-801 induced marked increases in locomotor activity and disruptions of circular swim maze acquisition that were to greater or lesser extents blocked or potentiated by neuroleptic compounds: SCH 23390 (0.005 and 0.05 mg/kg) and clozapine (5.0 and 10.0 mg/kg) dose-dependently antagonised MK-801 (0.3 mg/kg) induced locomotor activity whereas raclopride (0.1 mg/kg) and haloperidol (0.1 mg/kg) attenuated it dose-specifically. Amperozide (0.5 mg/kg) attenuated the MK-801 effect but potentiated it at the 2.0 mg/kg dose. In the circular swim maze, raclopride (0.01 mg/kg) and SCH 23390 (0.05 mg/kg) improved the acquisitive performance of rats administered MK-801 (0.03 mg/kg) acutely whereas clozapine (10.0 mg/kg) and amperozide (2.0 mg/kg) deteriorated the performance of MK-801-treated rats. Postnatal administration of MK-801 (0.05 mg/kg, day 11 after birth) induced severe functional alterations in adult mice. At 70 days of age, MK-801 mice showed an initial hypoactivity followed by marked hyperactivity in the motor activity test chambers. These mice showed deficits in habituation, a nonassociative form of learning. Their hyperactivity in the test chambers was reversed by a low dose of d-amphetamine (0.25 mg/kg). Taken together, these findings display a wide range of acute/long-term functional alterations induced by NMDA antagonists, particularly MK-801, associated with animal models of brain disorders.

Keywords: Keywords: MPTP; Hypokinesia; Dyskinesia; Locomotion; Rearing; Memantine; Amantadine; MK-801; L-Dopa; 5 mg/kg; Co-administration; Motor fluctuations; C57 BL/6 mice; Activity test cages; Hypoactivity; Hyperactivity; Wistar rats; Circular swim maze; Acquisition performance; Clozapine; Haloperidol; Amperozide; SCH 23390; Raclopride; Antagonism; Potentiation; Postnatal administration; Habituation; Neonatal; Adult; Rats; Mice

Glutamate-mediated striatal dysregulation and the pathogenesis of motor response complications in Parkinson's disease by J. D. Oh; T. N. Chase (pp. 133-139).

Chronically administered levodopa to Parkinson's disease (PD) patients ultimately produces alterations in motor response. Similarly, in 6-hydroxydopamine lesioned hemi-parkinsonian rats, chronic twice-daily administration of levodopa progressively shortens the duration of contralateral turning, an index of, the wearing-off fluctuations that occur in parkinsonian patients. The pathogenesis of these response alterations involves, in part, upregulation of corticostriatal glutamatergic synaptic transmission. Changes involving kinase and phosphatase signaling pathways within striatal dopaminoceptive medium-spiny neurons now appear to contribute to increased synaptic efficacy of glutamatergic receptors in these neurons. Glutamate-mediated striatal sensitization subsequently modifies basal ganglia output in ways that favor the appearance of parkinsonian motor complications. At the molecular level, transcriptional activation of striatal CREB and cdk5 may contribute to the persistent expression of these levodopa-induced response alterations. Conceivably, a safer and more effective therapy for PD can be provided by drugs that target signaling proteins within striatal spiny neurons or those that interact extracellularly with non-dopaminergic receptors such as AMPA and NMDA, adenosine, adrenergic, opioid, and serotonergic.

Keywords: Keywords: NMDA receptor; AMPA receptor; Medium spiny neuron; Phosphorylation; Signal transduction

Recognition and treatment of response fluctuations in Parkinson's disease: Review article by L. Verhagen Metman (pp. 141-145).

Patients with Parkinson's disease (PD) by definition benefit from treatment with the dopamine precursor levodopa. However, after 5 years of therapy 50% of patients experience motor response complications (MRC's): the benefit from each dose becomes shorter (wearing-off fluctuations), more unpredictable (on-off fluctuations) and associated with involuntary movements (dyskinesias). In addition these patients suffer from fluctuations in motor function that are inherent to the disease itself. Recent findings have lead to the suggestion that hyperfunction of NMDA receptors on striatal efferent neurons, as a consequence of chronic non-physiologic dopaminergic stimulation, contributes to the pathogenesis of MRC's. In PD patients blockade of striatal glutamate receptors with several NMDA-antagonists improve MRC's. With progression of PD the severity and complexity of MRC's magnify, obfuscating their pattern and their relation to the medication cycle. Only through detailed history taking and patient education will the physician be able to clarify the situation and establish a rational, targeted approach to the treatment of patients with advanced PD complicated by motor fluctuations and dyskinesias.

Keywords: Keywords: Motor response complications; Dyskinesias; Levodopa treatment

Glutamatergic mechanisms in different disease states: overview and therapeutical implications – An introduction by T. M. Tzschentke (pp. 147-152).

Glutamate is the most widely distributed excitatory transmitter in the central nervous system (CNS). It is acting via large – and still growing – families of receptors: NMDA-, AMPA-, kainate-, and metabotropic receptors. Glutamate has been implicated in a large number of CNS disorders, and it is hoped that novel glutamate receptor ligands offer new therapeutic possibilites in disease states such as chronic pain, stroke, epilepsy, depression, drug addiction and dependence or Parkinson's disease. While an extensive preclinical literature exists showing potential beneficial effects of NMDA-, AMPA-, kainate- and metabotropic receptor ligands, only NMDA receptor antagonists have been characterized clinically to any appreciable degree. In these trials it has been shown that while several compounds are therapeutically active, they also produce serious side effects at therapeutic doses. Current interest largely centers on the development of receptor subtype-selective compounds, namely compounds selective for receptors containing the NR2B subunit. Preclinical findings and the first clinical results are encouraging, and it may be that such subunit-selective compounds may have a sufficiently wide therapeutic window to be safe for human use.

Keywords: Keywords: Glutamate; NMDA receptor; AMPA receptor; Kainate receptor; Metabotropic receptor; NR2B; Animal model; Clinical trial; Pain; Stroke; Epilepsy; Depression; Anxiety; Addiction; Dependence; Withdrawal; Parkinson

Modulation of glutamate receptors: Strategies for the development of novel antidepressants by P. Skolnick (pp. 153-159).

On a biochemical level, conventional antidepressants have been shown to modulate synaptic levels of biogenic amines (i.e., serotonin, norepinephrine, and dopamine), most often by interfering with reuptake processes or inhibiting metabolism. Strategies directed at modulating glutamatergic transmission may overcome the principal limitations (i.e., delayed onset and low efficacy) that appear to be inherent to these conventional agents. In this brief overview, I summarize two glutamate-based approaches to develop novel antidepressants. These distinct and (on a cellular level) seemingly diametric strategies may converge on intracellular pathways that are also impacted upon by chronic treatment with biogenic amine based agents.

Keywords: Keywords: NMDA antagonists; Depression; Behavioral despair; AMPA; AMPA receptor potentiators

Cystine/glutamate exchange serves as the source for extracellular glutamate: Modifications by repeated cocaine administration by D. A. Baker; H. Shen; P. W. Kalivas (pp. 161-162).

Repeated administration of cocaine lowers the basal extracellular levels of glutamate in the nucleus accumbens as measured by microdialysis. The studies presented reveal that this long-term neuroadaptation elicited by repeated cocaine results from a decrease in the activity of cystine/glutamate exchange.

Keywords: Keywords: Cocaine; Cystine/glutamate exchanger; Microdialysis

The effects of NMDA receptor antagonists on acute morphine antinociception in mice by E. Kozela; P. Popik (pp. 163-168).

Antagonists of the N-methyl-d-aspartate (NMDA) receptor complex inhibit the development of tolerance to antinociceptive effects of morphine and upon acute administration, influence morphine antinociceptive activity. The analysis of numerous studies investigating acute interaction between NMDA receptor antagonists and morphine in mice indicate a variety of procedural differences and reveal that these compounds may potentiate, attenuate and produce no effect on morphine antinociception. The conditions responsible for such conflicting experimental outcome of acute interaction remain unclear. It appears that the effects of NMDA receptor antagonists on morphine tolerance are not causally related to their acute effects on morphine antinociception.

Keywords: Keywords: Opiates; Morphine; Antinociception; Pain; NMDA receptor antagonists; Glutamate

Novel approaches to targeting glutamate receptors for the treatment of chronic pain: Review article by B. A. Chizh (pp. 169-176).

Glutamatergic mechanisms are implicated in acute and chronic pain, and there is a great diversity of glutamate receptors that can be used as targets for novel analgesics. Some approaches, e.g. NMDA receptor antagonism, have been validated clinically, however, the central side-effects have remained the main problem with most compounds. Recently, some novel approaches have been explored as new compounds targeting some modulatory sites at the NMDA receptor (glycine_B and NR2B-subtype selective antagonists), as well as kainate and metabotropic glutamate receptors, have been discovered. Many of these compounds have demonstrated efficacy in animal models of chronic pain, and some of them appear to have a reduced side-effect liability compared to clinically tested NMDA antagonists. These recent advances are reviewed in the present work.

Keywords: Keywords: NMDA receptors; AMPA receptors; Kainate receptors; Metabotropic glutamate receptors; Inflammatory pain; Neuropathic pain

Formalin-induced changes of NMDA receptor subunit expression in the spinal cord of the rat by C. Gaunitz; A. Schüttler; C. Gillen; C. Allgaier (pp. 177-182).

Using RT-PCR, the present study investigated the effects of formalin administration on mRNA expression coding for NMDA receptor (NR) subunits and splice variants in rat lumbar spinal cord. Subsequent to formalin injection (5%; subcutaneously) into the hind paw of Sprague-Dawley rats, the animals exhibited the typical biphasic behavioural pain response. Spinal cord (L3-6) was prepared six hours after formalin injection. In controls, NR1-b predominated over NR1-a, and NR1-2 and NR1-4 exceeded over NR1-1 and NR1-3, respectively. Regarding the NR2 subunit expression in controls, NR2B exhibited the highest expression, followed by decreasing proportions of NR2C, NR2A, and NR2D. Formalin treatment did not affect NR1 splice variant expression but significantly increased and decreased the proportion of NR2A and NR2C, respectively. In summary, the present data demonstrate adaptive changes in the NR subunit expression pattern in rat spinal cord due to formalin injection.

Keywords: Keywords: Formalin; NMDA receptors; NR1 splice variants; NR2 subunits; Rat spinal cord; Pain

Neuro- and psychotropic effects of drugs active at metabotropic glutamate receptors – Therapeutic perspectives – Introduction by S. Wolfarth; K. Ossowska (pp. 183-184).

Localization and physiological roles of metabotropic glutamate receptors in the direct and indirect pathways of the basal ganglia by M. J. Marino; H. Awad; O. Poisik; M. Wittmann; P. J. Conn (pp. 185-191).

Our current understanding of the circuitry of the basal ganglia, and the pathophysiology of Parkinson's disease has led to major breakthroughs in the treatment of this debilitating movement disorder. Unfortunately, there are significant problems with the currently available pharmacological therapies that focus on dopamine replacement or dopaminergic agonists. Because of this, much effort has been focused on developing novel targets for the treatment of Parkinson's disease. The metabotropic glutamate receptors are a family of G-protein coupled receptors activated by glutamate. These receptors are differentially distributed throughout the basal ganglia in a manner suggesting that they may provide novel targets for the treatment of movement disorders. In this review we summarize anatomical and physiological data from our work and the work of other laboratories describing the distribution and physiological roles of metabotropic glutamate receptors in the basal ganglia with emphasis on possible therapeutic targets.

Keywords: Keywords: Basal ganglia; Metabotropic glutamate receptors; Movement disorders; Parkinson's disease

The role of striatal metabotropic glutamate receptors in Parkinson's disease by K. Ossowska; J. Konieczny; J. Wardas; K. Gołembiowska; S. Wolfarth; A. Pilc (pp. 193-198).

The primary cause of Parkinson's disease is a loss of dopamine in the corpus striatum. It has been postulated that this effect leads to disinhibition of the striopallidal pathway and secondarily, to a functional shift towards glutamatergic stimulation. The aim of the present study was to find out whether inhibition of glutamatergic transmission at a level of metabotropic glutamate receptors (mGluRs) in the striatum may alleviate parkinsonian-like symptoms in rats.The non-competitive antagonist of receptor subtype 5 (mGluR5), MPEP (1.0–10 mg/kg ip), or the agonist of group II mGluRs, LY354,740 (5–10 mg/kg ip), reduced haloperidol-induced muscle rigidity and catalepsy. Intrastriatal injections of the mGluR1 antagonist, (RS) AIDA (7.5–15 μg/0.5 μl), but not of the agonist of group II mGluRs, 2R,4R-APDC (7.5–15 μg/0.5 μl), inhibited the muscle rigidity induced by haloperidol.In order to search for an influence of mGluRs on the striopallidal pathway, the effect of MPEP or of the agonist of group II mGluRs, DCG-IV, on the proenkephalin (PENK) mRNA expression in the dorso-lateral striatum was examined by an in situ hybridization. Repeated MPEP (6 × 10 mg/kg ip) administration did not influence PENK expression in naïve rats, but diminished that increased by haloperidol. In contrast, repeated DCG-IV (3 × 1 nmol/4 μl icv) injections enhanced both the control and the haloperidol-increased levels of PENK expression.The obtained results suggest that blockade of group I mGluRs, or stimulation of group II mGluRs may be important to ameliorate parkinsonian symptoms. Striatal mGluRs may contribute to at least some of these effects.

Keywords: Keywords: Metabotropic glutamate receptors; Antiparkinsonian-like effects; Striatum

The role of striatal metabotropic glutamate receptors in degeneration of dopamine neurons: Review article by K. Gołembiowska; J. Konieczny; K. Ossowska; S. Wolfarth (pp. 199-205).

Degeneration of dopaminergic nigrostriatal neurons is a primary cause of Parkinson's disease. Oxidative stress, excitotoxicity and mitochondrial failure are thought to be key mechanisms resposible for degeneration of dopaminergic cells. We found that the selective antagonist of the mGluR5 subtype MPEP in a dose of 5 mg/kg diminshed basal and veratridine (100 μM)-stimulated dopamine release in rat striatum in an in vivo model of microdialysis. In contrast, MPEP given intrastriatally in a high concentration (500 μM) enhanced the striatal extracellular concentration of dopamine. DCG-IV (100 μM), a non-selective agonist of group II mGluRs, inhibited the veratridine-stimulated striatal dopamine release. In an animal model of neuroxicity in vivo, methamphetamine (5 × 10 mg/kg, injected at 2 h intervals) produced deficits in the striatal content of dopamine and its metabolites DOPAC and HVA 72 h after the treatment. MPEP (5 × 5 mg/kg) given before each methamphetamine injection reversed the decrease in the striatal content of dopamine and diminished the methamphetamine-induced dopamine outflow from nigrostriatal terminals. It is concluded that the MPEP-produced blockade of mGluR5 situated on dopaminergic cells, or the suppression of glutamate release in the subthalamic nucleus or substantia nigra pars reticulata may directly and indirectly cause a decrease in striatal dopamine release. However, inhibitory effect of DCG-IV on dopamine release can be induced by attenuation of excitatory input from corticostriatal terminals by activation of mGluR2/3. Regulation of dopamine carriers by MPEP, an antagonist of group I mGluRs may be responsible for the reversal of toxicity induced by methamphetamine.

Keywords: Keywords: Metabotropic glutamate receptors; Dopamine release; Neurotoxicity; Striatum

In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: Review article by R. Kuhn; A. Pagano; N. Stoehr; I. Vranesic; P. J. Flor; K. Lingenhöhl; W. Spooren; C. Gentsch; A. Vassout; A. Pilc; F. Gasparini (pp. 207-211).

There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain.We have characterized novel subtype-selective mGlu₅ receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu₅ receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety.

Keywords: Keywords: Group I metabotropic glutamate receptors; MPEP; SIB-1757; Anxiety; Pain

Antidepressant-like effect of MPEP, a potent, selective and systemically active mGlu5 receptor antagonist in the olfactory bulbectomized rats by J. M. Wieronska; B. Szewczyk; P. Branski; A. Palucha; A. Pilc (pp. 213-216).

Using the olfactory bulbectomy model of depression, we examined the antidepressant-like activity of 2-methyl-6-(phenylethynyl)-pyridine (MPEP) in rats. Bulbectomized rats required a significantly greater number of trials to acquire the response similar to sham-operated controls in the passive avoidance model. Both the prolonged (but not acute) treatment with MPEP and with antidepressant drug-desipramine restored the learning deficit. The results indicate that the prolonged blockade of mGlu5 receptors exerts antidepressant-like effects in rats.

Keywords: Keywords: Excitatory amino acids; Metabotropic glutamate receptors; mGlu5 receptors; MPEP; Antidepressant effects; Olfactory bulbectomy; Animal models of depression

Neurobiology of the CNS injury and repair: New roles of amino acids, growth factors and neuropeptides – Introduction by H. S. Sharma (pp. 217-219).

Role of creatine and phosphocreatine in neuronal protection from anoxic and ischemic damage by M. Balestrino; M. Lensman; M. Parodi; L. Perasso; R. Rebaudo; R. Melani; S. Polenov; A. Cupello (pp. 221-229).

Phosphocreatine can to some extent compensate for the lack of ATP synthesis that is caused in the brain by deprivation of oxygen or glucose. Treatment of in vitro rat hippocampal slices with creatine increases the neuronal store of phosphocreatine. In this way it increases the resistance of the tissue to anoxic or ischemic damage. In in vitro brain slices pretreatment with creatine delays anoxic depolarization (AD) and prevents the irreversible loss of evoked potentials that is caused by transient anoxia, although it seems so far not to be active against milder, not AD-mediated, damage. Although creatine crosses poorly the blood-brain barrier, its administration in vivo at high doses through the intracerebroventricular or the intraperitoneal way causes an increase of cerebral phosphocreatine that has been shown to be of therapeutic value in vitro. Accordingly, preliminary data show that creatine pretreatment decreases ischemic damage in vivo.

Keywords: Keywords: Creatine; Phosphocreatine; Ischemia; Anoxia; Stroke; Guanidinoacetate methyltransferase deficiency; Hyperornithinemia; Protection

Repeated topical application of growth hormone attenuates blood-spinal cord barrier permeability and edema formation following spinal cord injury by F. Nyberg; H. S. Sharma (pp. 231-239).

The neuroprotective efficacy of growth hormone on a focal spinal cord trauma induced alteration in the blood-spinal cord barrier (BSCB) and edema formation was examined in a rat model. Under Equithesin anaesthesia, one segment laminectomy was done over the T10–11 segments. Spinal cord injury was produced by making an incision into the right dorsal horn of the T10–11 segments (2 mm deep and 4 mm long). The animals were allowed to survive 5 h after injury. Highly purified rat growth hormone [rGH, 25 μl of a 1 μg/ml solution) was applied over 10 sec topically on the exposed surface of the spinal cord 30 min before injury. The identical doses of the rGH were repeated 0 min, 30 min, 60 min, 120 min, 180 min and 240 min following injury. Saline (0.9% NaCl) treated traumatised rats at identical intervals served as controls. Traumatised rats treated with saline exhibited marked edema formation and extravasation of Evans blue and ^[125]Iodine tracers in the spinal cord. At the ultrastructural level, perivascular edema and exudation of lanthanum across the endothelial cells was quite frequent in the spinal cord. Pretreatment with rGH significantly attenuated the edema formation and the extravasation of tracers in the spinal cord. In these rats, perivascular edema and infiltration of lanthanum across the endothelial cells was not much evident. These observations show that the rGH has the capacity to reduce the early manifestations of microvascular permeability disturbances and edema formation following trauma and further suggest a possible therapeutic potential of the hormone for the treatment of spinal cord injuries.

Keywords: Keywords: Blood-spinal cord barrier (BSCB); Edema; Laminectomy; Microvascular permeability; Rat; Rat growth hormone (rGH); Spinal cord injury; Topical application; Visual swelling

Role of VEGF in an experimental model of cortical micronecrosis by J. V. Lafuente; S. Bulnes; B. Mitre; H. H. Riese (pp. 241-245).

Vascular endothelial growth factor (VEGF) is a major mediator in angiogenesis and vascular permeability. In central nervous system (CNS) it plays a pivotal role as: 1. inductor of endothelial cell proliferation, migration and inhibition of apoptosis, and 2. mediator of vascular permeability and subsequently of brain edema. This ubiquitous epiphenomenon is a major complication in several CNS pathologies, including head trauma and stroke.After brain injury the expression of VEGF is increased contributing to disruption of the blood brain barrier (BBB). VEGF increase the permeability of BBB via the synthesis/release of nitric oxide and subsequent activation of soluble guanylate cyclase. The immunohistochemistry shows an increase of stained astrocytes and endothelial cells around cortical micronecrosis. VEGF immunopositivity distribution shows some correspondence with the blood brain barrier breakdown following a cortical micronecrosis.

Keywords: Keywords: VEGF; Cortical micronecrosis; Brain edema; BBB; Vascular endothelium

Nitric oxide synthase inhibitors in?uence dynorphin A (1–17) immunoreactivity in the rat brain following hyperthermia by H. S. Sharma; P. Alm (pp. 247-259).

The possibility that nitric oxide synthase (NOS) inhibitors influence dynorphin immunoreactivity following hyperthermia was examined in a rat model using a pharmacological approach. Previous reports from our laboratory show that hyperthermia induces an upregulation of NOS in several brain regions that seems to be instrumental in causing cell injury. Recent reports suggest that nitric oxide (NO) can influence dynorphin neurotransmission in the normal brain as well as in several pathological states. Since dynorphin is neurotoxic in different animal models of brain or spinal cord injury, it may be that the peptide will contribute to the cell injury in hyperthermia. The present investigation was carried out to determine whether hyperthermia can influence dynorphin immunoreactivity in the brain, and if so, whether inhibition of NOS will influence the peptide distribution in the brain following heat stress. Rats subjected to hyperthermia at 38°C for 4 h in a biological oxygen demand incubator (BOD) resulted in a marked upregulation of dynorphin immunoreactivity in several brain regions e.g., cerebral cortex, hippocampus, cerebellum and brain stem. Pretreatment of rats with two potent NOS inhibitors, L-NAME (30 mg/kg/day, i.p. for 7 days) or L-NMMA (35 mg/kg/day, i.p. for 7 days) significantly attenuated the dynorphin immunoreactivity in the brain. These drugs were also able to reduce hyperthermia induced blood-brain barrier (BBB) permeability, brain edema formation and cell injury. Taken together, our results suggest that (i) hyperthermia has the capacity to upregulate dynorphin immunoreactivity in the brain, (ii) inhibition of NOS considerably attenuates the dynorphin immunoreaction following heat stress and (iii) upregulation of dynorphin is somehow contributing to hyperthermia induced brain damage, not reported earlier.

Keywords: Keywords: Dynorphin; Hyperthermia; Brain injury; Immunoreactivity; Nitric oxide; Ultrastructure; L-NAME; L-NMMA; Nitric oxide synthase; Brain edema; Evans blue; [131]Iodine

A new antioxidant compound H-290/51 modulates glutamate and GABA immunoreactivity in the rat spinal cord following trauma by H. S. Sharma; P.-O. Sjöquist (pp. 261-272).

The involvement of the excitatory amino acid glutamate and the inhibitory amino acid gamma-amino butyric acid (GABA) in the pathophysiology of spinal cord injury is not known in details. This investigation is focused on the role of glutamate and GABA in a rat model of spinal cord trauma using immunohistochemistry. Spinal cord injury produced by a longitudinal incision of the right dorsal horn of the T10–11 segments resulted in profound edema and cell damage in the adjacent T9 segment at 5 h. Pretreatment with H-290/51 (50 mg/kg, p.o.), a potent antioxidant compound, effectively reduced the blood-spinal cord barrier (BSCB) permeability, edema formation and cell injury following trauma. At this time, untreated traumatised rats exhibited a marked increase in glutamate immunoreactivity along with a distinct decrease in GABA immunostaining in the T9 segment. These changes in glutamate and GABA immunoreactivity in traumatised rats were considerably attenuated by pretreatment with H-290/51. These results suggest that (i) oxidative stress contributes to alterations in glutamate and GABA in spinal cord injury, (ii) glutamate and GABA are important factors in the breakdown of the BSCB, edema formation and cell changes, and (iii) the antioxidant compound H-290/51 has a potential therapeutic value in the treatment of spinal cord injuries.

Keywords: Keywords: Glutamate; GABA; Spinal cord injury; Immunohistochemistry; Antioxidant; H-290/51; Ultrastructure; Edema; Blood-spinal cord barrier

Topical application of dynorphin A (1–17) antiserum attenuates trauma induced alterations in spinal cord evoked potentials, microvascular permeability disturbances, edema formation and cell injury by T. Winkler; H. S. Sharma; T. Gordh; R. D. Badgaiyan; E. Stålberg; J. Westman (pp. 273-281).

Dynorphin is a neuropeptide that is present in high quantities in the dorsal horn of the spinal cord. The peptide is actively involved in pain processing pathways. However, its involvement in spinal cord injury is not well known. Alteration in dynorphin immunoreactivity occurs following a focal trauma to the rat spinal cord. Infusion of dynorphin into the intrathecal space of the cord results in ischemia, cell damage and abnormal motor function. Antibodies to dynorphin when injected into the intrathecal space of the spinal cord following trauma improve motor recovery, reduce edema and cell changes. However, influence of dynorphin on trauma induced alteration in spinal cord bioelectrical activity is still not known. Spinal cord evoked potentials (SCEP) are good indicator of spinal cord pathology following trauma. Therefore, in present investigation, influence of dynorphin antibodies on trauma induced changes in SCEP were examined in our rat model. In addition, spinal cord edema formation, microvascular permeability disturbances and cell injury were also investigated. Our results show that topical application of dynorphin antiserum (1 : 200) two min before injury markedly attenuated the SCEP changes immediately after injury. In the antiserum treated animals, a significant reduction in the microvascular permeability, edema formation and cell injury was observed in the traumatised spinal cord. These observations suggest that (i) dynorphin is involved in the altered bioelectrical activity of the spinal cord following trauma, (ii) the peptide actively participates in the pathophysiological processes of cell injury in the spinal cord trauma, and (iii) the dynorphin antiserum has potential therapeutic value for the treatment of spinal cord injuries.

Keywords: Keywords: Amino acids; Dynorphin antiserum; Spinal cord injury; Edema; Spinal cord evoked potentials; Ultrastructure

Role of cholecystokinin-A and cholecystokinin-B receptors in anxiety by A. M. Hernandez-Gómez; R. Aguilar-Roblero; M. Pérez de la Mora (pp. 283-290).

Evidence from several laboratories indicates that the anxiogenic effects of cholecystokinin (CCK) are mediated by CCKB receptors. However, it has been reported that CCKA receptors have been found in brain and CCKA antagonists have anxiolytic properties. The aim of this work was to study whether CCKA receptors are also involved in the modulation of anxiety. Anxiogenic effects were observed in the elevated plus maze in rats when pure CCKB receptor agonists (CCK-4 and CCK-8 non-sulfated) or CCK-8S, a CCKB/CCKA agonist, were injected into the lateral ventricle. In contrast, CCK-33, a CCKA agonist or CCK-(1–21) and CCK-(26–29) were ineffective. Furthermore, the anxiogenic effects of CCK-8S were prevented by blocking CCKB but not CCKA receptors. Finally, CCK-33 injected into the postero-medial nucleus accumbens failed to affect the anxiety level of the rats. These results indicate that CCKA receptors are not involved in anxiety, as measured by the paradigms used in this work.

Keywords: Keywords: CCKA receptor; CCKB receptor; anxiety; elevated plus maze; CCK33

Molecular imaging of ischemia and angiogenesis – Introduction by O. Golubnitschaja (pp. 291-291).

Molecular imaging of perfusion disturbances in glaucoma by O. Golubnitschaja; K. Wunderlich; C. Decker; H. Mönkemann; H. H. Schild; J. Flammer (pp. 293-299).

Ocular ischemia resulting from perfusion disturbances may play a major role in initiation of glaucoma. Possibly secondary to ischemia autoimmunogenic events are activated in glaucoma patients with increased prevalence of systemic autoimmune diseases. The determination of potential molecular markers in blood leukocytes could be useful for early noninvasive diagnostics of glaucoma. Our study using subtractive hybridization showed altered gene expression in leukocytes of glaucoma patients in comparison to age and sex matched healthy subjects. Subtracted genes encoding lymphocyte IgE receptor (Fc epsilon RII/CD23), T cell-specific tyrosin kinase, thromboxan A2 receptor, alkaline phosphatase and Na⁺/K⁺-ATPase are differentially expressed in circulating leukocytes of glaucoma patients. These genes show expression profiles characteristic for adherent leukocytes which could be an important contributor to blood-brain barrier breakdown which has been found in glaucoma patients.

Keywords: Keywords: Molecular imaging; Perfusion disturbances; Vaso-spasm; Glaucoma; Gene hunting; Leukocytes

Targeting of MPEG-protected polyamino acid carrier to human E-selectin in vitro by H. W. Kang; R. Weissleder; A. Bogdanov, Jr. (pp. 301-308).

Targeted diagnostic agents are expected to have a significant impact in molecular imaging of cell-surface associated markers of proliferation, inflammation and angiogenesis. In this communication, we describe a new class of targeted polyamino acid-based protected graft copolymers (PGC) of poly-(L-lysine) and methyl poly-(ethylene glycol) (PGC) covalently conjugated with a monoclonal antibody fragment, F(ab′)₂. We utilized targeted PGC conjugates as carriers of near-infrared indocyanine fluorophores (Cy5.5) for optical imaging of endothelial cell populations expressing IL-1β inducible proinflammatory marker E-selectin. We compared two conjugation chemistries, involving either introduction of sulfhydryl group to F(ab′)₂, or via direct attachment of the antibody fragment directly to the chemically activated PGC. Both PGC-based targeted agents demonstrated high binding specificity (20–30 fold over non-specific uptake) and were utilized for imaging E-selectin expression on human endothelial cells activated with IL-1β.

Keywords: Keywords: E-selectin; Endothelium; IL-1β; Graft copolymer; Indocyanine; Antibody; Optical imaging

Perfusion and molecular diffusion-weighted MR imaging of the brain: In vivo assessment of tissue alteration in cerebral ischemia by S. Flacke; H. Urbach; W. Block; F. Träber; P. Mürtz; E. Keller; A. Hartmann; H. H. Schild (pp. 309-316).

The combined use of perfusion imaging (PI) and diffusion-weighted imaging (DWI) is opening a new window into the processes that occur during the first hours of ischemia. DWI detects changes in molecular diffusion associated with cytotoxic edema. PI characterizes the degree of regional hypoperfusion. Regions showing mismatches between DWI and PI, i.e. hypoperfused areas with normal diffusion behavior are considered potentially salvageable. We present results of 11 patients with an occlusion of the middle cerebral artery stem and spontaneous stroke evolution. Whereas the infarct was clearly visible on initial DWI and PI, surrounding tissue at risk of infarction was marked in all patients by an increased blood volume and transit time, but only in a subgroup (n = 3) where alteration were more pronounced this tissue at risk was progressively infarcted. These human DWI and PI data show alterations in the area of tissue at risk which correlates with infarct progression.

Keywords: Keywords: Diffusion-weighted imaging; Perfusion imaging; Stroke; Brain; Penumbra

In-vivo proton MR-spectroscopy of the human brain: Assessment of N-acetylaspartate (NAA) reduction as a marker for neurodegeneration by W. Block; F. Träber; S. Flacke; F. Jessen; C. Pohl; H. Schild (pp. 317-323).

Proton magnetic resonance spectroscopy (¹H-MRS) is a non-invasive method to investigate changes in brain metabolite composition in different cerebral diseases.We performed proton spectroscopy in patients with dementia of the Alzheimer's type (AD) and in patients with motor neuron disease (MND) with the aim to detect the specific metabolic pattern for these neurodegenerative disorders.In the MND group we found a significant reduction of NAA/tCr metabolite ratios in the motor cortex, which correlates with the disease severity and the clinical lateralization of neurological symptoms and further decreases in the time course of the disease. In AD patients a reduction of NAA/tCr was observed in the medial temporal lobe.Since NAA is exclusively expressed in neurons as shown by immunohistochemical studies, reduced NAA levels suggest neuronal loss or dysfunction in the observed regions.The observed regional metabolic alterations reflect the neuronal basis of the characteristic neurological symptoms in AD (dementia) and MND (muscular palsy) and mirrors the disease progress over time.

Keywords: Key words: Proton MR spectroscopy; Neurodegeneration; N-Acetylaspartate; Alzheimer's disease; Motor neuron disease

Effects of cytoskeletal modi?cations on Ca²⁺ in?ux after cerebral ischemia by K. B. Fink; M. Paehr; P. C. Djoufack; C. Weissbrich; J. Bösel; M. Endres (pp. 325-329).

The fungal toxin cytochalasin D as well as endogenous gelsolin depolymerize filamentous actin which may induce dynamic uncoupling of membrane ion channels. In vitro application of cytochalasin D reduced NMDA-induced [³H]noradrenaline release from mouse brain neocortical slices by 38%. In gsn deficient neocortical synaptosomes [Ca²⁺]_i increase in response to K⁺ (30 mM) depolarization was 33% higher than in wild-type. After transient focal cerebral ischemia K⁺-induced [Ca²⁺]_i increase in neocortical synaptosomes was 56% lower than in synaptosomes prepared from the non-ischemic contralateral hemisphere. After in vivo pretreatment with cytochalasin D 10 min before MCA occlusion K⁺-induced [Ca²⁺]_i increase in synaptosomes in vitro prepared 1 h after reperfusion from the ischemic hemisphere was only 25% lower than in contralateral synaptosomes, while cytochalasin D pretreatment in vivo did not reduce K⁺-induced [Ca²⁺]_i increase in vitro. Hence, presynaptic Ca²⁺ influx and subsequently neuronal vulnerability are attenuated by increased and are aggravated by decreased F-actin depolymerization.

Keywords: Keywords: Voltage-gated Calcium channels; F-actin; Stroke; Cytochalasin D

Early molecular events in the development of the diabetic cardiomyopathy by H. Mönkemann; A. S. De Vriese; H. J. Blom; L. A. J. Kluijtmans; S. G. Heil; H. H. Schild; O. Golubnitschaja (pp. 331-336).

Oxidative damage to DNA has been well documented in cardiac cells isolated from diabetic patients and rats with streptozotocin-induced diabetes mellitus (DM). This study evaluates possible molecular mechanisms for early events in the development of DM-induced cardiomyopathy. Methods: To analyze the mechanism of overexpression of p21^WAF1/CIP1 and inhibition of cyclin D₁ expression in cardiomyocytes of diabetic rats we examined the methylation status of these genes by MS-PCR and assessed the possibility of epigenetic control of their expression. Results: We found that the steady-state expression of both genes is influenced by their methylation status, as an epigenetic event, of their 5′-flanking regions upon development of diabetes. Conclusions: Oxidative damage contributes to the development of cardiomyopathy via p53-dependent activation of cardiac cell death. This pathway includes de novo methylation of the P53-inducible p21^WAF1/CIP1-gene encoding a protein which binds to and inhibits a broad range of cyclin-cyclin-dependent kinase complexes.

Keywords: Keywords: Diabetes mellitus; Cardiomyopathy; p21WAF1/CIP1; Cyclin D1; CpG Methylation; Gene regulation

Effects of dietary de?ciency of selective amino acids on the function of the cornea and lens in rats by A. Wegener; O. Golubnitschaja; W. Breipohl; H. H. Schild; G. F. J. M Vrensen (pp. 337-342).

Effects of dietary deficiencies of tryptophan and methionin on the transparency of cornea and lens were investigated in young rats (Brown-Norway, BN; Sprague-Dawley, SD) over 3 months. Transparency of the cornea and lens were evaluated in weekly intervals using a photo-slitlamp microscope. After sacrifice and lens fresh weight determination the lenses were prepared for histopathology. Methionin deficiency had no effect on the parameters investigated. Tryptophan deficiency caused severe loss of body weight in both strains, with additional loss of hair in SD rats. These developed corneal neovascularisations and cataracts. BN rats showed an enhanced zone of discontinuity in the lens. Diet intermission arrested the pathological processes in the eye which restarted when feeding the diet again. This observation is supported by lens fresh weight data. DNA staining evidenced that tryptophan deficiency arrested lens fiber maturation in both strains but stimulated corneal neovascularisation only in SD rats.

Keywords: Keywords: Tryptophan/methionin deficiency; Corneal/lens transparency; Cataract; Albino / pigmented rats; Fiber cell maturation

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.23, #1-3)