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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.19, #1)
Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures by J. Zimmer; B. W. Kristensen; B. Jakobsen; J. Noraberg (pp. 7-21).
Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the induced neurodegeneration, we have developed standardized protocols, including – a) densitometric measurements of the cellular uptake of propidium iodide (PI), – b) histological staining by Flouro-Jade, – c) lactate dehydrogenase (LDH) release to the culture medium, – d) immunostaining for microtubulin-associated protein 2, and – e) general and specific neuronal and glial cell stains. The results show good correlation between the different markers, and are in accordance with results obtained in vivo. Examples presented in this review will focus on the use of PI uptake to monitor the excitotoxic effects of – a) KA and AMPA (and NMDA) in hippocampal slice cultures, and – b) KA and AMPA in corticostriatal slice cocultures, with demonstration of differentiated neuroprotective effects of NBQX in relation to cortex and striatum and KA and AMPA. A second set of studies include modulation of hippocampal KA-induced excitotoxicity and KA-glutamate receptor subunit mRNA expression after long-term exposure to low, non-toxic doses of KA and NBQX.We conclude that organotypic brain slice cultures, combined with standardized procedures for quantitation of cell damage and receptor subunit changes is of great potential use for studies of excitotoxic, glutamate receptor-induced neuronal cell death, receptor modulation and related neuroprotection.
Keywords: Keywords: Amino acids; Brain slice cultures; Hippocampus; Striatum; Cerebral cortex; Kainic acid; AMPA; NBQX; Propidium iodide
Margatoxin and iberiotoxin, two selective potassium channel inhibitors, induce c-fos like protein and mRNA in rat organotypic dorsal striatal slices by A. Saria; H. S. Fischer; C. Humpel; A. Pfattner; D. S. Schatz; R. Schuligoi (pp. 23-31).
The isolated single organotypic slice model allows to investigate the effects of drugs and toxins on the expression of transcription factors in the striatum without dopaminergic and glutamatergic interactions. In this study the effects of margatoxin and iberiotoxin on the expression of c-fos mRNA by in situ hybridization as well as on c-fos like protein by immunohistochemistry in isolated dorsal striatum after 10 days in culture were investigated. C-fos mRNA dose-dependently increased 30 min after incubation with margatoxin and iberiotoxin. Expression of c-fos like protein was transiently detected 3 h afterwards. This effect is independent from extrinsic neuronal circuitry as dopamine neurons were found to be absent in the cultured slices. It is concluded that inhibition of voltage-gated as well as calcium-activated (Slo) potassium channels leads to activation of gene transcription in striatal neurons which may trigger long-term changes in transmitter plasticity.
Keywords: Keywords: Amino acids; Scorpion toxins; Striatum; Gene expression
Excitatory amino acids, monoamine, and nitric oxide synthase systems in organotypic cultures: biochemical and immunohistochemical analysis by M. Herrera-Marschitz; C. Kohlhauser; S. Gomez-Urquijo; R. Ubink; M. Goiny; T. Hökfelt (pp. 33-43).
The nigrostriatal and mesolimbic systems of the rat have been re-constructed using the organotypic culture model, whereby neonatal brain tissue is grown in vitro for approximately one month. The nigrostriatal cultures consisted of tissue from the substantia nigra, dorsal striatum and frontoparietal cortex; while the mesolimbic cultures included the ventral tegmental area, ventral striatum and cingulate cortex.The cultures were grown at 35°C in normal atmosphere, using a tube-roller device placed in a cell incubator and changing the medium every 3–4 days. The in vitro development was evaluated with an inverted microscope equipped with a variable relief contrast function. Samples were taken directly from the medium in the culture tube and analysed for several amino acids with HPLC. After a month the cultures were fixed and processed for immunohistochemistry.High levels of glutamate and aspartate were observed every time the medium was changed, but the levels rapidly decreased reaching a steady state after approximately 24 h. A decrease in the levels was also observed along development, reaching stable values (∼2 μM and ∼0.12 μM for glutamate and aspartate, respectively) at approximately two weeks, but only when the cultures showed an apparently healthy development. The levels were approximately 10 times higher in deteriorating or apparently damaged cultures. Glutamine levels were in the mM range and remained stable along the entire experiment. No differences were observed among nigrostriatal and mesolimbic cultures.Immunohistochemistry confirmed the impressions obtained from microscopic and biochemical analysis along the in vitro development, revealing apparently healthy neuronal systems with characteristics similar to those observed in vivo, when tyrosine hydroxylase and nitric oxide synthase, markers for dopamine and nitric oxide containing neurons, respectively, were analysed. In the substantia nigra, nitric oxide synthase-positive networks surrounded tyrosine hydroxylase-positive neurons, while in the striatum nitric oxide synthase dendrites were surrounded by tyrosine hydroxylase-positive nerve terminals, suggesting a reciprocal interaction among dopamine and nitric oxide containing neurons.Thus, the organotypic model appears to capture many of the neurochemical and morphological features seen in vivo, providing a valuable model for studying in detail the neurocircuitries of the brain.
Keywords: Keywords: Amino acids; Basal ganglia; Dopamine; Nitric oxide; Excitatory amino acids; Organotypic culture; Immunohistochemistry
Dale's principle and glutamate corelease from ventral midbrain dopamine neurons by D. Sulzer; S. Rayport (pp. 45-52).
While direct application of dopamine modulates postsynaptic activity, electrical stimulation of dopamine neurons typically evokes excitation. Most of this excitation appears to be due to activation of collateral pathways; however, several lines of evidence have suggested that there is a monosynaptic component due to glutamate corelease by dopamine neurons. Recently, more direct evidence obtained in culture has shown that ventral midbrain dopamine neurons release both dopamine and glutamate. Moreover, they appear to do so from separate release sites, calling into question recent modifications of Dale's Principle. The neurochemical phenotype of a given synapse may be determined by subcellular neurotransmitter levels, uptake, or storage. However, the relationship between dopamine and glutamate release from dopamine neuron synapses in the intact brain – and the mechanisms involved – has yet to be resolved.
Keywords: Keywords: Amino acids; Autapses; VMAT2; Substantia nigra; Ventral tegmental area; Phosphate-activated glutaminase; Tyrosine hydroxylase
Diversity of glutamate transporter expression and function in the mammalian retina by T. Rauen (pp. 53-62).
Glutamate is the major excitatory neurotransmitter of the mammalian retina and glutamate uptake is essential for normal transmission at glutamatergic synapses.Between photoreceptors and second order neurons, increases in light intensity are signaled by decreases in the concentration of glutamate within the synaptic cleft. In such a system the precise control of glutamate in the synaptic cleft is thus essential and glutamate transporters are thought to contribute to this process. As demonstrated here, all neuronal and macroglial cells of the retina appear to express high-affinity glutamate transporters. GLAST1, GLT1, EAAC1 and EAAT5 are expressed in the retina and exhibit unique localisation and functional properties. In the present study we summarize retinal glutamate transporter expression, identify the major glutamate uptake site in the mammalian retina and discuss the possible functional roles of different glutamate transporter subtypes in glutamatergic neurotranmission in the retina.
Keywords: Keywords: Amino acids; Glutamate; Transporter; Retina; Expression; Transmission
Lack of coupling between GABA release and GABA synthesis in the rat brain via GABAB autoreceptors by A. Aguilar-García; B. González-Frankenberger; T. Ramón-Frías; B. J. Méndez-Franco; M. Pérez de la Mora (pp. 63-77).
GABA is synthesized within GABA terminals through a highly compartmentalized process in which glial-derived glutamine is a major precursor and its release is modulated by GABAB autoreceptors. The aim of this work was to ascertain whether or not GABA synthesis and release are coupled in the rat brain through a GABAB autoreceptor-mediated modulation. It was found that (−)baclofen (30 μM) reduces the K+ stimulated release of [3H]GABA in synaptosomes and prisms (10 μM) from cerebral cortex, while at the same concentrations (−)baclofen failed to modify the synthesis of [3H]GABA from [3H]glutamine in cortical and hypothalamic slices, prisms and in cortical synaptosomes. In this latter preparation, identical results were observed when (−)baclofen was added to Krebs-Tris media, containing 5 or 15 mM K+ concentration. In agreement with these latter results, glutamic acid decarboxylase (GAD) activity from cortical and hypothalamic prisms was not affected by 1–100 μM (−)baclofen. Similar results on GABA synthesis were also observed when 1–100 μM 3-aminopropil(methyl)-phosphinic acid or GABA was used instead of (−)baclofen to stimulate GABAB autoreceptors. [3H]GABA release, [3H]GABA synthesis from [3H]glutamine and GAD activity were also insensitive to the action of the GABAB antagonist CGP 52432 (10–100 μM). Likewise, muscimol (0.3–100 μM) did not affect GABA synthesis. Our results indicate that unlike GABA release, GABA synthesis is not modulated by GABAB autoreceptors.
Keywords: Keywords: Amino acids; GABA-synthesis; GABA-release; GABA; (; )Baclofen; GABAB autoreceptors; CGP 52432; Rat brain
Stimulation of group II metabotropic glutamate receptors or inhibition of group I ones exerts anxiolytic-like effects in rats by A. Pilc; E. Chojnacka-Wójcik; E. Tatarczyńska; J. Borycz; B. Kroczka (pp. 81-86).
Using the conflict drinking Vogel test in rats as a model we examined the anxiolytic-like activity of (S)-4-carboxyphenylglycine (S-4CPG), an antagonist of group I metabotropic glutamate receptors (mGlu receptors), of (RS)-α-methylserine-O-phosphate-monophenyl ester (MSOPPE), an antagonist of group II mGlu receptors, and of (2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (L-CCG-I), an agonist of group II mGlu receptors. The obtained results indicate that intrahippocampal administra-tion of S-4CPG and L-CCG-I, but not MSOPPE to rats produces a dose-dependent anticonflict effect, which is unrelated to the reduced perception of the stimulus or to an increased thirst drive. The hippocampus may be one of the neuroanatomical sites of the anxiolytic-like effects of either agent.
Keywords: Keywords: Amino acids; Metabotropic glutamate receptors; (S)-4-carboxyphenylglycine (S-4CPG); (2S; 1′S; 2′S)-2-(carboxycyclopropyl) glycine (L-CCG-I); Drinking conflict test; Intrahippocampal injection
The role of glutamate receptors in antipsychotic drug action by K. Ossowska; M. Pietraszek; J. Wardas; G. Nowak; W. Zajaczkowski; S. Wolfarth; A. Pilc (pp. 87-94).
It has recently been postulated that disturbances in glutamatergic neurotransmission may contribute to the pathophysiology of schizophrenia. Therefore the aim of the present study was to evaluate the role of glutamate NMDA and group II metabotropic receptors in the antipsychotic drug action. To this aim the influence of some well-known neuroleptics on cortical NMDA receptors was examined. Furthermore, their behavioral effects were compared with those of the novel agonist of group II glutamate metabotropic receptors, LY 354740, in some animal models of schizophrenic deficits. We found that long-term administration of the typical neuroleptic haloperidol and the atypical one clozapine increased the number of NMDA receptors labelled with [3H]CGP 39653 in different cortical areas. Long-, but not short-term, treatment with haloperidol and raclopride diminished the deficit of prepulse inhibition produced by phencyclidine, which is a model of sensorimotor gating deficit in schizophrenia. In contrast, neither short- nor long-term treatment with clozapine influenced the phencyclidine effect in that model. Acute treatment with LY 354740 reversed neither (1) the deficit of prepulse inhibition produced by phencyclidine or apomorphine, nor (2) the impairment in a delayed alternation task induced by MK-801, which is commonly used to model the frontal lobe deficits associated with schizophrenia. The present study suggests that an increase in the density of cortical NMDA receptors may be important to a longterm neuroleptic therapy. Conversely, the results do not support the role of group II metabotropic glutamate receptors in the antipsychotic drug action.
Keywords: Keywords: Amino acids; NMDA receptors; Group II metabotropic glutamate receptors; Cerebral cortex; Neuroleptics; Prepulse inhibition; Delayed alternation task
The role of metabotropic glutamate receptor (mGluR) ligands in parkinsonian muscle rigidity by S. Wolfarth; J. Konieczny; E. Lorenc-Koci; K. Ossowska; A. Pilc (pp. 95-101).
It has been shown that the primary striatal dopaminergic hypofunction which is at the origin of Parkinson's disease, results in a secondary hyperactivity of glutamatergic neurotransmission. In the search for a therapy of Parkinson's disease, ionotropic, mainly NMDA, receptor antagonists were found to have moderately beneficial, yet also some undesirable side-effects. Therefore the present study was aimed at determining whether some metabotropic glutamate receptor (mGluR) ligands may have antiparkinsonian effects in the haloperidol-induced muscle rigidity. To this end three mGluR ligands were used: the potent and selective mGluR I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), the mixed group II agonist/group I antagonist (S)-4-carboxy-3-hydroxyphenyl-glycine ((S)-4-C3HPG), and the potent group II agonist (+)-2-aminobicyclo[3.1.0.]hexane-2,6,-dicarboxylic acid (LY354740). Only LY354740 penetrated the brain from the periphery; for this reason other drugs were injected bilaterally into the rostral striatum or nucleus accumbens. The muscle tone was recorded by a mechanomyographic/electromyographic (MMG/EMG) method which measured the resistance of a rat's hind foot and the EMG reflex response of its muscles to passive movements. (S)-4C3HPG (5 and 15 μg/0.5 μl) and LY354740 (5 and 10 mg/kg i.p.) diminished the muscle rigidity induced by haloperidol (1 mg/kg i.p.). AIDA (0.5–15 μg/0.5 μl) injected into the striatum was only slightly effective in the highest dose used. However, when injected into the nucleus accumbens AIDA (15 μg/0.5 μl) significantly and strongly counteracted the haloperidol-induced muscle rigidity. Our results suggest that stimulation of group II striatal mGluRs seems to play a major role in diminution of parkinsonian-like muscle rigidity. However, it seems that the antagonism of group I mGluRs located in the nucleus accumbens may also be of importance to the antiparkinsonian effect.
Keywords: Keywords: Amino acids; Parkinsonian-like muscle rigidity; Ionotropic glutamate receptors; Metabotropic glutamate receptors; Striatum
On the role of inhibitory glutamate receptors in N-methyl-D-aspartate- and dopamine-receptor mediated motor behavior of rats by U. O. Kronthaler; W. J. Schmidt (pp. 103-118).
The physiological function of inhibitory group II metabotropic glutamate-receptors, a family of second messenger coupled glutamate-receptors, for motor behavior is almost unknown. The aim of this study is to address this topic by quantifying motor effects of the preferential group II agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine, administered i.c.v. (62.5, 125.0, 187.5, 250.0, 500.0 nmol/4 μl), in an open-field equipped with a hole-board. (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine decreased spontaneous locomotor and exploratory behavior, which was blocked be the group II antagonist (2S)-alpha-ethylglutamic acid (250.0 nmol/4 μl). Locomotion induced by the N-methyl-D-aspartate-receptor antagonist dizocilpine (0.08, 0.16, 0.32 mg/kg) was counteracted by the group II agonist (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine, but an antagonism towards dizocilpine did not occur in all aspects of motor behavior evaluated. In contrast to the antagonism of dizocilpine induced locomotion, D,L-amphetamine (1.0, 2.0, 3.0 mg/kg) induced locomotion was not antagonised by (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine.The results suggest that group II agonists may be devoid of psychotomimetic effects in humans and even may antagonise this side effect of N-methyl-D-aspartate receptor antagonists. Since group II activation and N-methyl-D-aspartate-receptor blockade very efficiently protects against excitotoxic neurodegeneration, selective group II agonists may allow novel pharmacotherapeutical approaches in pathophysiological conditions characterised by a glutamatergic hyperactivity, like epilepsy, ischemia and trauma.
Keywords: Keywords: Amino acids; Basal ganglia; Neuroprotection; Schizophrenia; Metabotropic Group II
Cell-type specificity of mGluR activation in striatal neuronal subtypes by A. Pisani; G. Bernardi; P. Bonsi; D. Centonze; P. Giacomini; P. Calabresi (pp. 119-129).
The effects of metabotropic glutamate receptor (mGluR) activation were studied in medium spiny neurons and large aspiny (LA) interneurons by means of electrophysiological and optical recordings. DCG-IV and L-SOP, agonists for group II and III mGluRs, respectively, produced a presynaptic inhibitory effect on corticostriatal glutamatergic excitatory postsynaptic potentials (EPSPs) in both spiny and LA cells. Activation of group I mGluRs by the selective agonist 3,5-DHPG produced no effect on membrane properties and glutamatergic transmission in spiny neurons, whereas it did cause a membrane depolarization in LA interneurons coupled to increased input resistance. In combined optical and electrophysiological experiments, in spiny neurons 3,5-DHPG enhanced membrane depolarization and intracellular calcium (Ca2+) levels induced by NMDA applications, but not in LA interneurons. These data suggest the existence of a positive interaction between NMDA and group I mGlu receptors only in medium spiny cells which might, at least partially, account for the differential vulnerability to excitotoxic damage observed in striatal neuronal subtypes.
Keywords: Keywords: Amino acids; Striatum; Electrophysiology; Metabotropic glutamate receptors; Excitotoxicity
Low affinity channel blocking (uncompetitive) NMDA receptor antagonists as therapeutic agents – toward an understanding of their favorable tolerability by M. A. Rogawski (pp. 133-149).
Studies in experimental models have suggested that NMDA receptor antagonists may have utility in the treatment of a wide variety of neurological and psychiatric disorders. However, clinical trials have not been encouraging largely because the antagonists evaluated to date have exhibited unacceptable neurobehavioral side effects. In animals, therapeutic doses of some low-affinity channel blocking (uncompetitive) NMDA receptor antagonists are associated with less gross neurological impairment and behavioral toxicity than other types of NMDA receptor antagonists. Favorable clinical experiences with several such agents has bolstered confidence in the neurotherapeutic potential of low affinity NMDA antagonists. This article reviews current research attempting to explain the improved tolerability of such antagonists. While no single mechanism appears to account for the reduced toxicity of such agents, kinetic properties, particularly rapid blocking rate, seem to be of key importance. Other factors include partial trapping, reduced agonist-independent (closed channel) block, subunit selectivity (particularly for receptors that do not contain the NR2A subunit), combined block at allosteric (voltage-independent) sites, and synergistic therapeutic effects produced by additional actions at receptor targets apart from NMDA receptors (e.g., weak positive allosteric modulation of GABAA receptors or state-dependent Na+ channel block).
Keywords: Keywords: Amino acids; NMDA-Receptors antagonists
Low affinity use-dependent NMDA receptor antagonists show promise for clinical development by G. C. Palmer; D. Widzowski (pp. 151-155).
The success of the low affinity use-dependent NMDA receptor antagonists to reach clinical trials can be readily attributed to their wider margins of safety and lack of neurotoxicity at higher doses. Several mechanistic differences distinguish the low affinity from the high affinity use-dependent antagonists: 1) Differential regional affinities for the various NMDA receptor subtypes; 2) The static receptor blockade due to the faster on/off rate receptor kinetics which limit, but do not totally prevent the amount of Ca+2 entry into the cell during glutamate-induced depolarization; and 3) Rapid egress of the compounds from the ion channel during recovery resulting in less membrane trapping between transmission pulses. Advanced clinical trials are in progress for the following indications: epilepsy, stroke, head trauma, tardive dyskinesia, pain plus Parkinson's, Huntington's and Alzheimer's diseases.
Keywords: Keywords: Amino acids; NMDA; Low affinity use-dependent antagonists; Clinical status; Epilepsy; Stroke; Head trauma; Neurodegeneration
Memantine and the amino-alkyl-cyclohexane MRZ 2/579 are moderate affinity uncompetitive NMDA receptor antagonists –in vitro characterisation by C. G. Parsons; W. Danysz; G. Quack (pp. 157-166).
There is general agreement that moderate affinity uncompetitive NMDA receptor antagonists combine good efficacy and tolerability in animal models of disturbances in glutamatergic transmission. There are several theories on which properties are important for this profile including 1, rapid access to the channel at the start of pathological overactivity 2, rapid, voltage-dependent relief of blockade during physiological synaptic activation and 3, partial untrapping. Merz has developed a series of novel uncompetitive NMDA receptor antagonists based on the cyclohexane structure. In cultured hippocampal neurones MRZ 2/579 (1-amino-1,3,3,5,5-pentamethyl-cyclohexane) shows similar blocking kinetics to memantine (Kon 10.7 * 104 M−1 sec−1, Koff 0.20 sec−1 at −70 mV) and binds at the same depth in the NMDA receptor channel (δ = 0.8). The potency of MRZ 2/579 assessed as Kd = Koff/Kon = 1.87 μM agrees well with the IC50 of 1.29 μM against steady-state currents in cultured hippocampal neurones (at −70 mV) and with the Ki in [3H]-MK-801 binding of 0.65 μM. MRZ 2/579 protected cultured cortical neurones against glutamate toxicity with an IC50 of 2.16 μM and was also effective in protecting hippocampal slices against hypoxia / hypoglycaemia-induced reduction of fEPSP amplitude in CA1 with an EC50 of 7.01 μM. MRZ 2/579 has similar potency and bio-availability to memantine in vivo assessed using microdialysis, microiontophoresis and MES-induced seizures. Initial characterization in animal models provides strong support for the assump-tion that MRZ 2/579 could be a useful therapeutic in morphine/alcohol dependence, inhibition of morphine tolerance, chronic pain and as a neuroprotective agent.
Keywords: Keywords: Amino acids; N-Methyl-D-aspartate; Uncompetitive antagonist; Memantine; MRZ 2/579
NMDA channel blockers: memantine and amino-aklylcyclohexanes –In vivo characterization by W. Danysz; C. G. Parsons; G. Quack (pp. 167-172).
The previous overviews provided the basis for better therapeutic efficacy/tolerability of low to moderate affinity NMDA channel blockers. This prediction finds support in in vitro studies comparing protective and plasticity impairing effects (therapeutic vs. side-effect) of memantine and (+)MK-801. In fact it turned out that memantine had a far better therapeutic index. But can it be confirmed in the in vivo situation?
Keywords: Keywords: Amino acids; NMDA; Channel blockers; Memantine
Differences in degree of trapping between AR-R15896 and other uncompetitive NMDA receptor antagonists by T. H. Lanthorn; G. A. R. Mealing; P. Morley (pp. 173-175).
NMDA antagonists like AR-R15896 have been selected on the basis of their good therapeutic indices. As Dr. Rogawski has pointed out, there may be a number of molecular factors which can improve the therapeutic index of NMDA antagonists. In this paper we will consider three factors; use-dependence, low affinity/fast kinetics, and partial trapping.
Keywords: Keywords: Amino acids; NMDA; Use-dependence; Partial trapping
NPS 1506, a moderate affinity uncompetitive NMDA receptor antagonist: preclinical summary and clinical experience by A. L. Mueller; L. D. Artman; M. F. Balandrin; E. Brady; Y. Chien; E. G. DelMar; A. Kierstead; T. B. Marriott; S. T. Moe; J. L. Raszkiewicz; B. VanWagenen; D. Wells (pp. 177-179).
NPS Pharmaceuticals, Inc. (NPS) has synthesized a series of open-channel blockers with varying potencies at the NMDA receptor. NPS 1506 (Fig. 1) is a moderate affinity antagonist that inhibits NMDA/glycine-induced increases in cytosolic calcium in cultured rat cerebellar granule cells (IC50 = 476 nM) and displaces the binding of [3H]MK-801 to rat cortical membranes (IC50 = 664 nM).
Keywords: Keywords: Amino acids; Glutamate; Neuroprotection; NMDA receptors
Dopaminergic denervation enhances susceptibility to hydroxyl radicals in rat neostriatum by R. M. Kostrzewa; J. P. Kostrzewa; R. Brus (pp. 183-199).
To determine if greater amounts of hydroxyl radical (•OH) are formed by dopamine (DA) denervation and treatment with L-dihydroxyphenylalanine (L-DOPA), the neostriatum was DA denervated (99% reduction in DA content) by 6-hydroxydopamine treatment (134 μg icv, desipramine pretreatment) of neonatal rats. At 10 weeks the peripherally restricted dopa decarboxylase inhibitor carbidopa (12.5 mg/kg i.p.) was administered 30 min before vehicle, L-DOPA (60 mg/kg i.p.), or the known generator of reactive oxygen species, 6-hydroxydopa (6-OHDOPA) (60 mg/kg i.p.); and this was followed 30 min later (and 15 min before termination) by the spin trap, salicylic acid (8 μmoles icv). By means of a high performance liquid chromatographic method with electrochemical detection, we found a 4-fold increase in the non-enzymatically formed spin trap product, 2,3-dihydroxybenzoic acid (2,3-DHBA), with neither L-DOPA nor 6-OHDOPA having an effect on 2,3-DHBA content of the neostriatum. Basal content of 2,5-DHBA, the enzymatically formed spin trap product, was 4-fold higher vs. 2,3-DHBA in the neostriatum of untreated rats, while L-DOPA and 6-OHDOPA each reduced formation of 2,5-DHBA. We conclude that DA innervation normally suppresses •OH formation, and that the antiparkinsonian drug L-DOPA has no effect (2,3-DHBA) or slightly reduces (2,5-DHBA) •OH formation in the neostriatum, probably by virtue of its bathing the system of newly formed •OH.
Keywords: Keywords: Amino acids; Denervation; Dopamine; 6-Hydroxydopa; 6-Hydroxydopamine; Hydroxyl radicals; Levodopa; Parkinson's disease; Reactive oxygen species
Functional aspects of the ventral pallidum by B. D. Kretschmer (pp. 201-210).
The ventral pallidum is part of the corticoaccumbo-thalamocortical loop of the basal ganglia. In the past the function of this structure was discussed as a pure relay station in the process of limbic-motor integration. Some recent studies, however, underline that on the level of the ventral pallidum motor behavior can be modulated. The stimulation and inhibition of the different transmitter systems that converge in the ventral pallidum (dopamine, glutamate, GABA, neuropeptides) have implications in repetitive-, disinhibited-, learning- and reinforced behavior. The present review summarizes available data of these parameter related to this behavior, i.e. locomotion, reward-related behavior, prepulse inhibition, memory and neurochemistry.
Keywords: Keywords: Amino acids; Ventral pallidum; Corticoaccumbo-thal-amocortical loop; Locomotion; Reward; Prepulse inhibition; Memory; Microdialysis
The medial prefrontal cortex as a part of the brain reward system by T. M. Tzschentke (pp. 211-219).
This review will briefly summarize experimental evidence for an involvement of the medial prefrontal cortex (mPFC) in reward-related mechanisms in the rat brain. The mPFC is part of the mesocorticolimbic dopaminergic system. It receives prominent dopaminergic input from the ventral tegmental area (VTA) and, via the mediodorsal thalamus, inputs from other subcortical basal ganglia structures. In turn it projects back to the VTA and the nucleus accumbens septi (NAS), which are generally considered as main components of the brain reward system.Evidence for the involvement of the mPFC in reward-related mechanisms comes mainly from three types of studies, conditioned place preference (CPP), intracranial self-stimulation (ICSS), and self-administration. Work will be summarized that has shown that certain drugs injected into the mPFC can produce CPP or that lesions of the mPFC can disrupt the development of CPP, that ICSS is obtained with the stimulating electrode placed in the mPFC, and that certain drugs are self-administered into the mPFC or that lesions of the mPFC disrupt the peripheral self-administration of certain drugs.However, it has also been shown that the role of the mPFC in reward is not uniform. For example, the mPFC appears to be particularly important for the rewarding actions of cocaine, while it appears not to be important for the rewarding actions of amphetamine. Also, different subareas of the mPFC appear to be differentially involved in the rewarding actions of different drugs.Taken together, the available evidence shows that some drugs can produce reward directly within the mPFC, and that some drugs, even though not having direct rewarding effects within the mPFC, depend on the function of the mPFC for the mediation of their rewarding effects.
Keywords: Keywords: Amino acids; Medial prefrontal cortex; Reward; Conditioned place preference (CPP); Intracranial self-stimulation (ICSS); Self-administration
Atypical antipsychotic-like effect of AMPA receptor antagonists in the rat by T. H. Svensson; J. M. Mathé (pp. 221-226).
Systemic administration of two chemically different AMPA receptor antagonists, GYKI52466, 20 mg/kg, and LY326325, 18 mg/kg, given subcutaneously, caused a selective suppression of conditioned avoidance response in the rat with preservation of escape behavior. The number of intertrial crosses was not affected and no catalepsy was observed. These experimental results indicate, in principle, an antipsychotic effect of AMPA receptor antagonists with a low liability for extrapyramidal side effects and, consequently, a pharmacological profile consonant with atypical antipsychotic drugs.
Keywords: Keywords: Amino acids; Conditioned avoidance response; AMPA; Kainate; Behavior; Antipsychotic
Effects of kainic acid lesioning on poly(ADP-ribose) polymerase (PARP) activity in the rat striatum in vivo by C. Cosi; E. Cavalieri; A. Carcereri de Prati; M. Marien; H. Suzuki (pp. 229-237).
Poly(ADP-ribose) polymerase (PARP) is activated in glutamate-induced toxicity of neurons in culture (Cosi et al., 1994). Since injection of the excitatory amino acid, kainic acid (KA) into the rat striatum induces a delayed neuronal death, the effects of this in vivo excitotoxin lesioning procedure on striatal PARP activity was investigated. PARP activity was measured in striatal extracts both in the absence ("endogenous" activity) and presence ("total" activity) of exogenously-added fragmented DNA. KA (5 nmols/1 μl) produced significant and time-dependent changes in striatal PARP activity, compared to saline-injected control animals: no changes at 6 h after intrastriatal KA, a 68% and 48% decrease in endogenous and total PARP activity respectively at 12 h, a doubling in endogenous PARP activity at 24 h, and a 382% and 60% increase in endogenous and total activities at 1 week after KA. PARP cleavage was not detected at any time point. These results suggest a participation of PARP in KA-induced toxicity in the brain in vivo.
Keywords: Keywords: Amino acids; Kainic acid; Neurodegeneration; Excitotoxicity; Poly(ADP-ribose) polymerase; PARP; In vivo
Drug treatments to reduce excitotoxicity in vivo: a potential for α 2-adrenoceptor antagonists? by J.-C. Martel; P. Chopin; F. Colpaert; M. Marien (pp. 239-252).
It is hypothesized that the locus coeruleus-noradrenergic system controls compensatory and repair mechanisms in the CNS, and that its dysfunction is a critical factor in the progression of central neurodegenerative diseases. Pharmacological activation of locus coeruleus neurons can be achieved with α 2-adrenoceptor antagonists, and such compounds are protective in vivo in some models of brain injury where excitotoxicity is thought to be a causative factor. To further explore this neuroprotective potential, the effects of a 7-day treatment with the α 2-antagonists, (+)-efaroxan and (±)-idazoxan, were evaluated in rats undergoing a unilateral lesioning of the striatum with the excitotoxin, quinolinic acid. The α 2-antagonist treatments reduced both the ipsiversive circling response to apomorphine and the deficit of choline acetyltransferase in the lesioned animals. To elucidate the mechanisms underlying this neuroprotective effect, a modulation of the extracellular levels of amino acids within the striatum was investigated using in vivo microdialysis. Intrastriatal injection of quinolinic acid increased taurine and tyrosine levels by 2–2.5 fold, while most other amino acids were not significantly altered; the effect of (+)-efaroxan on these changes is being investigated. Further research is required to identify which of several possible mechanisms is involved in the neuroprotective action of α 2-antagonists in vivo.
Keywords: Keywords: Amino acids; Excitotoxicity; Quinolinic acid; Intracerebral microdialysis; α2-Adrenoceptor antagonists; Neuroprotection
Induction of p53 in the glutamate-induced cell death program by D. Uberti; M. Grilli; M. Memo (pp. 253-261).
Fifteen minute exposure of primary cultures of cerebellar granule cells to micromolar concentrations of glutamate results in apoptotic cell death. Among the intracellular events triggered by glutamate, we identified two transcriptional factors, i.e. the p50 member of the NF-κB family and the tumor suppressor phosphoprotein p53, that are apparently linked by a sequential trascriptional program. We found that pretreatment of the cultures with aspirin (ASA), which inhibits NF-κB activation, resulted in a complete prevention of glutamate-induced p53 immunoreactivity. The same results were obtained pretreating the cells with a specific p53 antisense oligonucleotide. Both ASA and p53 antisense abolished glutamate-induced apoptosis. We also found that two other proteins, the cyclin dependent kinase inhibitor p21 and DNA mismatches repair MSH2, whose encoding genes are well known target of p53, were upregulated by glutamate. On these bases, we propose NF-κB, p53, p21 and MSH2 as relevant contributors of the glutamate-induced pro-apoptotic pathway.
Keywords: Keywords: Amino acids; Cerebellar neurons; Cyclin-dependent kinase; Excitotoxicity; Glutamate; Neurodegenerative diseases; Tumour suppressor gene
NMDA receptor dependent and independent components of veratridine toxicity in cultured cerebellar neurons are prevented by nanomolar concentrations of terfenadine by R. Díaz-Trelles; A. Novelli; G. Puia; M. Baraldi; M. T. Fernández-Sánchez (pp. 263-272).
Exposure of cultured neurons to nanomolar concentrations of terfenadine prevented the NMDA receptor-mediated early appearance (30 min.) of toxicity signs induced by the voltage sensitive sodium channel activator veratridine. Terfenadine also provided an histamine-insensitive protection against delayed neurotoxicity by veratridine (24 h), occurring independently of NMDA receptor activation, while not protecting from excitotoxicity following direct exposure of neurons to glutamate. Terfenadine reduced tetrodotoxin-sensitive inward currents, and reduced intracellular cGMP formation following veratridine exposure.Our data suggest that nanomolar concentrations of TEF may reduce excitatory aminoacid release following neuronal depolarization via a presynaptic mechanism involving voltage sensitive sodium channels, and therefore may be considered as a prototype for therapeutic drugs in the treatment of diseases that involve excitatory aminoacid neurotransmission.
Keywords: Keywords: Amino acids; Glutamate release; Excitotoxicity; Terfenadine; Sodium channels
Possible mediation of quinolinic acid-induced hippocampal damage by reactive oxygen species by T. W. Stone; W. M. H. Behan; M. MacDonald; L. G. Darlington (pp. 275-281).
Several differences exist between quinolinic acid and N-methyl-D-aspartate (NMDA) in the potency and pharmacology of their neurotoxic actions in the brain, suggesting that quinolinic acid may act by mechanisms additional to the activation of NMDA receptors, possibly involving lipid peroxidation. In the present review, studies are considered which have attempted to determine whether free radicals might contribute to the neuronal damage induced by quinolinic acid. Following Injections into the hippocampus of anaesthetised rats, quinolinic acid induced damage is prevented by melatonin, by an action not blocked by the melatonin receptor blocker luzindole. Deprenyl, but not the non-selective monoamine oxidase inhibitor nialamide, also prevent quinolinic acid-induced damage. In vitro, seversl groups have shown that quinolinic acid can induce lipid peroxidation of brain tissue The results suggest that free radical formation contributes significantly to quinolinic acid-induced damage in vivo.
Keywords: Keywords: Amino acids; Quinolinic acid; Kynurenines; Melatonin; Deprenyl; Antioxidants; Free radicals; Reactive oxygen species; Neuroprotection; Neurotoxicity; Excitotoxicity
Electrophysiological effects of exogenous and endogenous kynurenic acid in the rat brain: studies in vivo and in vitro by H. E. Scharfman; J. H. Goodman; R. Schwarcz (pp. 283-297).
In this review, recent studies on the electrophysiological effects of de novo synthesized ("endogenous") kynurenic acid (KYNA) are discussed. Endogenous KYNA is normally formed as a byproduct of tryptophan metabolism. Evidence for a physiological role in neuronal excitability has not been strong, in part because brain levels are much lower than the KD of KYNA at the glycine site of the NMDA receptor, where KYNA is thought to exert its most potent effect. The results suggest that, unexpectedly, even low concentrations of endogenous KYNA have physiological consequences. These levels of KYNA reduced the number of hippocampal slices with spontaneous epileptiform discharges after exposure to buffer lacking magnesium. However, effects on evoked responses to single afferent stimuli were not detected. Taken together, the data argue for a potentially important role of endogenous KYNA in suppression of seizure-like activity, and suggest a novel approach to anticonvulsant drug development that could have few side effects.
Keywords: Keywords: Amino acids; Anticonvulsant; Entorhinal cortex; Glycine; Hippocampus; N-Methyl-D-aspartate; Tryptophan
In vivo assessment of kynurenate neuroprotective potency and quinolinate excitotoxicity by T. P. Obrenovitch; J. Urenjak (pp. 299-309).
Three complementary questions related to the kynurenine pathway and excitotoxicity were addressed in this study: (i) Which extracellular levels of quinolinic acid (QUIN) may be neurotoxic? (ii) Which extracellular levels of kynurenic acid (KYNA) may control excessive NMDA-receptor function? (iii) Can "anti-excitotoxic" levels of KYNA be reached by inhibition of kynurenine-3-hydroxylase (i.e. inhibition of QUIN synthesis and shunts of kynurenine metabolism toward KYNA)? Multifunctional microdialysis probes were used in halothane anaesthetised rats to apply NMDA or QUIN directly to the brain, with or without co-perfusion of KYNA, to record the resulting local depolarisations, and to monitor changes in dialysate KYNA after kynurenine-3-hydroxylase inhibition. QUIN produced concentration-dependent depolarisations with an estimated EC50 (i.e. concentration in the perfusion medium) of 1.22 mM. The estimated ED50 for KYNA inhibition of NMDA-responses was 181 μM. Kynurenine-3-hydroxylase inhibition (Ro-61-8048, 100 mg/kg i.p.) increased dialysate KYNA 11 times (to 33.8 nM) but without any reduction of NMDA-responses. These data challenge the notion that extracellular accumulation of endogenous QUIN may contribute to excessive NMDA-receptor activation in some neurological disorders, and the suitability of kynurenine-3-hydroxylase inhibition as an effective anti-excitotoxic strategy.
Keywords: Keywords: Amino acids; Kynurenine pathway; Kynurenic acid; Quinolinic acid; NMDA-receptor; Excitotoxicity; Microdialysis
Perinatal kynurenine pathway metabolism in the normal and asphyctic rat brain by G. Ceresoli-Borroni; R. Schwarcz (pp. 311-323).
The kynurenine pathway of tryptophan degradation contains several metabolites which may influence brain physiology and pathophysiology. The brain content of one of these compounds, kynurenic acid (KYNA), decreases precipitously around the time of birth, possibly to avoid deleterious N-methyl-D-aspartate (NMDA) receptor blockade during the perinatal period. The present study was designed to determine the levels of KYNA, the free radical generator 3-hydroxykynurenine (3-HK), and their common precursor L-kynurenine (L-KYN) between gestational day 16 and adulthood in rat brain and liver. The cerebral activities of the biosynthetic enzymes of KYNA and 3-HK, kynurenine aminotransferases (KATs) I and II and kynurenine 3-hydroxylase, respectively, were measured at the same ages. Additional studies were performed to assess whether and to what extent kynurenines in the immature brain derive from the mother, and to examine the short-term effects of birth asphyxia on brain KYNA and 3-HK levels. The results revealed that 1) the brain and liver content of L-KYN, KYNA and 3-HK is far higher pre-term than postnatally; 2) KAT I and kynurenine 3-hydroxylase activities are quite uniform between E-16 and adulthood, whereas KAT II activity rises sharply after postnatal day 14; 3) during the perinatal period, KYNA, but not L-KYN, may originate in part from the maternal circulation; and 4) oxygen deprivation at birth affects the brain content of both KYNA and 3-HK 1 h but not 24 h later.
Keywords: Keywords: Amino acids; Asphyxia; Excitotoxicity; 3-Hydroxykynurenine; Kynurenic acid; N-Methyl-D-aspartate (NMDA)
Anticonvulsant effects of the 3-hydroxyanthranilic acid dioxygenase inhibitor NCR-631 by J. Luthman (pp. 325-334).
The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the pro-convulsive excitotoxin quinolinic acid. In the present study, the anticonvulsant effect of the 3-HAO inhibitor NCR-631 was investigated in models of chemically- and sound-induced seizures. Administration of NCR-631 i.c.v. at a dose of 300 nmol in Sprague-Dawley rats was found to prolong the latency of occurrence of pentylenetetrazole (PTZ)-induced seizures. Also systemic pre-treatment with NCR-631 s.c. in N.M.R.I. mice subjected to PTZ-induced seizures provided an increase in the latency until onset of seizures, concomitant with a reduction in the severity of the seizures. However, the anticonvulsant effect of NCR-631 was short lasting (15–30 min), and only observed at a dose of 250 mg/kg. A similar dose- and time-dependent anticonvulsant effect of NCR-631 was found in seizure-prone DBA/2J mice following sound-induced convulsions. Hence, the findings show that NCR-631 has anticonvulsant properties against generalized tonic-clonic seizures of different origin, suggesting that it may constitute a useful tool to study the role of kynurenines in various convulsive states.
Keywords: Keywords: Amino acids Seizures; Epilepsy; Kynurenines; Quinolinic acid
Antioxidant compounds EGB-761 and BN-520 21 attenuate heat shock protein (HSP 72 kD) response, edema and cell changes following hyperthermic brain injury by J. Westman; K. Drieu; H. S. Sharma (pp. 339-350).
Influence of the extract of Gingko biloba (EGB-761) and one of its constituent Gingkolide B (BN-52021) on hyperthermia induced cellular damage and heat shock protein (HSP 72 kD) response was examined in a rat model. Rats subjected to 4 h heat stress at 38°C in a biological oxygen demand (BOD) incubator (relative humidity 50–55%, wind velocity 20–25 cm/sec) resulted in profound edema and cell injury in many parts of the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus and brain stem. Immunostaining of HSP 72 kD showed marked upregulation in the damaged and distorted neurons located within the edematous area. Pretreatment with EGB-761 (50 mg/kg/day, p.o.) and BN-520 21 (2 mg/kg, p.o.) per day for 5 days significantly reduced HSP expression and attenuated cell damage. Our results show that EGB-761 and its component Gingkolide B (BN-52021) has the capacity to reduce edema and cell injury following hyperthermia and this effect of the compound is somehow associated with a reduction in cellular stress response as evidenced with a reduction in HSP expression.
Keywords: Keywords: Amino acids; Hyperthermia; Heat stress; Heat shock protein (HSP 72 kD); Edema; Cell injury; Antioxidants; EGB-761-BN 52021
Neurotrophic factors influence upregulation of constitutive isoform of heme oxygenase and cellular stress response in the spinal cord following trauma by H. S. Sharma; F. Nyberg; T. Gordh; P. Alm; J. Westman (pp. 351-361).
The influence of brain derived neurotrophic factor (BDNF) or insulin like growth factor-1 (IGF-1) on spinal cord trauma induced carbon monoxide (CO) production and cellular stress response was examined using immunostaining of the constitutive isoform of the hemeoxygenase (HO-2) enzyme and the heat shock protein (HSP 72 kD) expression in a rat model. Subjection of rats to a 5 h spinal trauma inflicted by an incision into the right dorsal horn at T10–11 segment markedly upregulated the HO-2 and HSP expression in the adjacent spinal cord segments (T9 and T12). Pretreatment with BDNF or IGF-1 significantly attenuated the trauma induced HSP expression. The upregulation of HO-2 was also considerably reduced. These results show that BDNF and IGF-1 attenuate cellular stress response and production of CO following spinal cord injury which seems to be the key factors in neurotrophins induced neuroprotection.
Keywords: Keywords: Amino acids; Spinal cord injury; Heme oxygenase; Heat shock protein; Carbon monoxide; Growth factors; BDNF; IGF-1; Immunohistochemistry; Cell injury; Spinal cord edema
Growth hormone attenuates alterations in spinal cord evoked potentials and cell injury following trauma to the rat spinal cord by T. Winkler; H. S. Sharma; E. Stålberg1; R. D. Badgaiyan; J. Westman; F. Nyberg (pp. 363-371).
The influence of exogenous rat growth hormone on spinal cord injury induced alterations in spinal cord evoked potentials (SCEP) and edema formation was examined in a rat model. Repeated topical application of rat growth hormone (20 μl of 1 μg/ml solution) applied 30 min before injuryand at 0 min (at the time of injury), 10 min, 30 min, 60 min, 120 min, 180 min, and 240 min, resulted in a marked preservation of SCEP amplitude after injury. In addition, the treated traumatised cord showed significantly less edema and cell changes. These observations suggest that growth hormone has the capacity to improve spinal cord conduction and attenuate edema formation and cell injury in the cord indicating a potential therapeutic implication of this peptide in spinal cord injuries.
Keywords: Keywords: Amino acids; Growth hormone; Spinal cord injury; Edema formation; Spinal cord evoked potentials; Spinal cord edema; Cell injury
Spinal nerve lesion induces upregulation of constitutive isoform of heme oxygenase in the spinal cord by T. Gordh; H. S. Sharma; M. Azizi; P. Alm; J. Westman (pp. 373-381).
The influence of carbon monoxide (CO) on chronic spinal nerve lesion induced spinal cord neurodegeneration was examined using immunohistochemical expression of the constitutive isoform of its synthesising enzyme, hemeoxygenase-2 (HO-2) in a rat model. Spinal nerve lesion at L-5 and L-6 level was produced according to the Chung model of neuropathic pain and rats were allowed to survive for 8 weeks. Sham operated rats, in which the spinal nerves were exposed but not ligated, served as controls. Ligation of spinal nerves in rats resulted in an upregulation of HO-2 expression which was most pronounced in the ipsilateral gray matter of the spinal cord compared to the contralateral side. In these rats, morphological investigations showed distorted neurons, membrane disruption, synaptic damage and myelin vesiculation. Sham operated rats did not show an upregulation of HO-2 expression and the structural changes in the spinal cord were absent. These observations strongly suggest that spinal nerve lesion is associated with an increased production of CO which is somehow contributing to the neurodegenerative changes in the spinal cord, not reported earlier.
Keywords: Keywords: Amino acids; Nerve lesion; Neuropathic pain; Heme oxygenase; Carbon monoxide; Cell injury; Immunohistochemistry
A new antioxidant compound H-290/51 attenuates nitric oxide synthase and heme oxygenase expression following hyperthermic brain injury by P. Alm; H. S. Sharma; P.-O. Sjöquist; J. Westman (pp. 383-394).
Influence of a new anti-oxidant compound H-290/51 on expression of nitric oxide synthase (NOS) and heme oxygenase (HO) enzymes responsible for nitric oxide (NO) and carbon monoxide (CO) production, respectively was examined in the CNS following heat stress in relation to cell injury. Exposure of rats to 4 h heat stress at 38°C in a biological oxygen demand (BOD) incubator (relative humidity 50–55%, wind velocity 20–25 cm/sec) resulted in profound edema and cell injury in many parts of the cerebral cortex, hippocampus, cerebellum, thalamus, hypothalamus and brain stem. Immunostaining of constitutive isoforms of neuronal NOS (nNOS) and HO-2 revealed marked upregulation in damaged and distorted neurons located within the edematous brain regions. Pretreatment with H-290/51 (50 mg/kg, p.o., 30 min before heat stress) significantly reduced the edematous swelling and cell injury and resulted in a marked attenuation of nNOS and HO-2 expression. These observations suggest that upregulation of NOS and HO is associated with cell injury, and the antioxidant compound H-290/51 is neuroprotective in heat stress.
Keywords: Keywords: Amino acids; Hyperthermia; Heat stress; Brain edema; Nitric oxide synthase; Heme oxygenase; Oxidative stress; H-290/51; Cell injury