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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.14, #1-3)

Satellite on neurobiology by W. J. Schmidt; M. Herrera-Marschitz (pp. 1-2).

Glutamate and other neurotransmitters in control of motor behaviour by C. S. Wolfarth (pp. 3-3).

Dopamine-glutamate interactions in the basal ganglia by Prof. Dr. W. J. Schmidt (pp. 5-10).

In an attempt to formulate a working hypothesis of basal-ganglia functions, arguments are considered suggesting that the basal ganglia are involved in a process of response selection i.e. in the facilitation of “wanted” and in the suppression of “unwanted” behaviour. The meso-accumbal dopamine-system is considered to mediate natural and drug-induced reward and sensitization. The meso-striatal dopamine-system seems to fulfill similar funcions: It may mediate reinforcement which strengthens a given behaviour when elicited subsequently, but which is not experienced as reward or hedonia.Glutamate as the transmitter of the corticofugal projections to the basal ganglia nuclei and of the subthalamic neurons is critically involved in basal ganglia funcions and dysfunctions; for example Parkinson's disease can be considered to be a secondary hyperglutamatergic disease. Additionally, glutamate is an essential factor in the plasticity response of the basal-ganglia. However, opposite to previous suggestions, the NMDA-receptor blocker MK-801 does not prevent psychostimulant- nor morphine-induced day to day increase (sensitization) of locomotion. Also the day to day increase of haloperidol-induced catalepsy was not prevented by MK-801.

Keywords: Basal ganglia loops; Reward; Sensitization; NMDA receptor; Parkinson's disease

The role of striatal glutamate receptors in models of Parkinson's disease by K. Ossowska; E. Lorenc-Koci; J. Konieczny; S. Wolfarth (pp. 11-15).

The aim of the study was to examine the effect of antagonists of the NMDA receptor on the parkinsonian-like muscle rigidity in rats. Reserpine and haloperidol increased the muscle resistance of the hind foot to passive movements, as well as the reflex electromyographic (EMG) activity in the gastrocnemius and tibialis anterior muscles. MK-801 (0.32-1.28 mg/kg sc), an uncompetitive antagonist of the NMDA receptor, and L-701,324 (5-40 mg/ kg ip), an antagonist of the glycine site, reduced the muscle tone and the reflex EMG activity enhanced by reserpine or haloperidol. AP-5 (2 and 5 ,μg/ 0.5 pl), a competitive antagonist of the NMDA receptor, and 5,7-dichlorokynurenic acid (1.0-4.5μg/0.5 pl), the glycine site antagonist injected bilaterally into the rostral striatum, inhibited the muscle rigidity induced by haloperidol. In contrast, AP-5, injected alone bilaterally into the intermediate-caudal striatum induced muscle rigidity. The present results suggest that: (1) the inhibitory effect of the NMDA receptor antagonists on the parkinsonian-like muscle rigidity depends, at least partly, on their action on the rostral striatum; (2) the blockade of NMDA receptors in the intermediate-caudal striatum may reduce the beneficial impact of these compounds.

Keywords: Parkinsonian-like muscle rigidity; NMDA receptor antagonists; Striatum

The role of NMDA receptors in the slow neuronal degeneration of Parkinson's disease by L. D. Loopuijt; W. J. Schmidt (pp. 17-23).

Parkinson's disease is a disorder, in which neurons of various neuronal systems degenerate. Furthermore, in such degenerating neurons, the cytoskeleton seems to be affected. In this respect, Parkinson's disease resembles Alzheimer's disease. Since it has been shown, that elevated levels of intracellular calcium can disrupt the cytoskeleton and that the stimulation of glutamate (NMDA) receptors can cause high intracellular concentrations of calcium, it has been suggested, that the stimulation of glutamate receptors plays a role in the slow degeneration in Alzheimer's and Parkinson's disease. In case of the degeneration of the dopaminergic nigrostriatal system in Parkinson's disease, neurons that contain calcium binding protein appear to be less vulnerable than the neurons that lack it, suggesting that calcium binding protein might protect these neurons from degeneration by preventing that cytosolic calcium concentrations increase excessively. And, since there is in the nigrostriatal system a glutamatergic afferent pathway (the prefrontonigral projection) and since dopaminergic nigrostriatal neurons contain postsynaptic NMDA receptors, glutamatergic excitation may play a role in the degeneration of the nigrostriatal system in Parkinson's disease. If so, it may be possible to protect the neurodegeneration of these dopaminergic neurons by NMDA receptor antagonists.

Keywords: NMDA receptors; Excitotoxicity; Chronic neuronal degeneration; Nigrostriatal neurons; Parkinson's disease

Modulation of motor behaviour by NMDA- and cholecystokinin-antagonism by Dr. C. Broberger; D. Blacker; L. Giménez-Llort; M. Herrera-Marschitz; S. -O. Ögren; T. Hükfelt (pp. 25-31).

Motor behaviour relies on complex neurochemical interactions in the basal ganglia, in particular the striatum. Antagonistic influences in this region are exerted by afferent projections from, on the one hand, the ventral mesencephalon, utilizing dopamine as a transmitter, and, on the other hand, from the cerebral cortex, signalling by the excitatory amino acid glutamate. The activity in both these neuronal populations appears to be regulated by the neuropeptide cholecystokinin. This article concentrates on interactions between cholecystokinin and glutamate, summarizing some recent morphological, biochemical and behavioural findings. It is suggested that cholecystokinin, acting via the cholecystokininB receptor, potentiates the glutamatergic excitatory input to the striatum.

Keywords: Cerebral cortex; Glutamate; Nucleus accumbens; Schizophrenia; Striatum

Adenosine A_2A receptors inhibit the conductance of NMDA receptor channels in rat neostriatal neurons by W. N örenberg; K. Wirkner; H. Aßmann; M. Richter; Dr. P. Illes (pp. 33-39).

Whole-cell patch clamp experiments were carried out in rat striatal brain slices. In a subset of striatal neurons (70–80%), NMDA-induced inward currents were inhibited by the adenosine AZA receptor selective agonist CGS 21680. The non-selective adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline and the AZA receptor selective antagonist 8-(3chlorostyryl) caffeine abolished the inhibitory action of CGS 21680. Intracellular GDP-β-S, which is known to prevent G protein-mediated reactions, also eliminated the effect of CGS 21680. Extracellular dibutyryl cAMP, a membrane permeable analogue of cAMP, and intracellular Sp-cAMPS, an activator of cAMP-dependent protein kinases (PKA), both abolished the CGS 21680-induced inhibition. By contrast, Rp-cAMPS and PKI 14–24 amide, two inhibitors of PKA had no effect. Intracellular U-73122 (a phospholipase C inhibitor) and heparin (an inositoltriphosphate antagonist) prevented the effect of CGS 21680. Finally, a more efficient buffering of intracellular Ca²⁺ by a substitution of EGTA (11 mM) by BAPTA (5.5 mM) acted like U-73122 or heparin. Hence, AZA receptors appear to negatively modulate NMDA receptor channel conductance via the phospholipase C/inositoltriphosphate/Ca²⁺ pathway rather than the adenylate cyclase/PKA pathway.

Keywords: Rat striatum; Medium spiny neuron; Adenosine AZA receptor; NMDA receptor channel; Whole-cell patch clamp

Antagonists of glutamate in the treatment of Parkinson's disease: From laboratory to the clinic by M. S. Starr (pp. 41-42).

Effects of glutamate antagonists on the activity of aromatic L-amino acid decarboxylase by A. Fisher; C. S. Biggs; Dr. M. S. Starr (pp. 43-49).

This study examines the hypothesis that glutamate tonically suppresses the activity of the enzyme aromatic L-amino acid decarboxylase (AADC), and hence the biosynthesis of dopamine, to explain how antagonists of glutamate receptors might potentiale the motor actions of L-DOPA in animal models of Parkinson's disease. A variety of glutamate antagonists were therefore administered acutely to normal rats, which were sacrificed 30–60 min later and AADC activity assayed in the substantia nigra pars reticulate (SNr) and corpus striatum (CS). The NMDA receptor-ion channel antagonists MK 801, budipine, amantadine, memantine and dextromethorphan all caused a pronounced in creased in AADC activity, more especially in the SNr than CS. The NMDA glycine site antagonist (R)-HA 966 produced a modest increase in AADC activity in the CS but not SNr, whilst the NMDA polyamine site antagonist eliprodil, the NMDA competitive antagonist CGP 40116 and the AMPA antagonist NBQX were without effect. The results suggest that an increase in dopamine synthesis might contribute to the L-DOPA-facilitating actions of some glutamate antagonists.

Keywords: L-DOPA; Aromatic L-amino acid decarboxylase; Glutamate antagonists; Substantia nigra; Corpus striatum

Role of excitatory amino acids in the ventral tegmental area for central actions of non-competitive NMDA-receptor antagonists and nicotine by Prof. T. H. Svensson; J. M. Mathé; G. G. Nomikos; B. Schilström (pp. 51-56).

The putative role of non-NMDA excitatory amino acid (EAA) receptors in the ventral tegmental area (VTA) for the increase in dopamine (DA) release in the nucleus acumbens (NAC) and the behavioural stimulation induced by systemically administered dizocilpine (MK-801) was investigated. Microdialysis was utilized in rats with probes in the VTA and NAC. The VTA was perfused with the AMPA and kainate receptor antagonist CNQX (0.3 or 1.0 mM) or vehicle and dialysates from the NAC were analyzed with high-performance liquid chromatography for DA. Forty min after onset of CNQX or vehicle perfusion of the VTA MK-801 (0.1 mg/kg) was injected subcutaneously (sc). Subsequently, typical MK-801 induced behaviours were assessed. The MK-801 induced hyperlocomotion was associated with a 50% increase of DA levels in NAC dialysates. Both the MK-801 evoked hyperlocomotion and DA release in the NAC were effectively antagonized by CNQX perfusion of the VTA. However, by itself the CNQX or vehicle perusion of the VTA did not affect DA levels in NAC or the rated behaviours. The results indicate that MK-801 induced hyperlocomotion and increased DA release in the NAC are largely elicited within the VTA via activation of non-NMDA EAA receptors, tentatively caused by locally increased EAA release. In contrast, the enhanced DA output in the NAC induced by systemic nicotine (0.5 mg/kg sc) was not antagonized by intra VTA infusion of CNQX (0.3 or 1.0 mM), but instead by infusion of the NMDA receptor antagonist AP-5 (0.3 or 1.0 mM) into the VTA, which by itself did not alter DA levels in the NAC. Thus, the probably indirect, EAA mediated activation of the mesolimbic DA neurons in the VTA by MK-801 and nicotine, respectively, seems to be mediated via different glutamate receptor subtypes.

Keywords: MK-801; Locomotion; Dopamine; Ventral tegmental area; Glutamate receptors

Role of excitatory amino acids in the regulation of dopamine synthesis and release in the neostriatum by M. J. Zigmond; S. L. Castro; K. A. Keefe; E. D. Abercrombie; A. F. Sved (pp. 57-62).

We have explored the role of excitatory amino acids in the increased dopamine (DA) release that occurs in the neostriatum during stress-induced behavioral activation. Studies were performed in awake, freely moving rats, usingin vivo microdialysis. Extracellular DA was used as a measure of DA release; extracellular 3,4-dihydroxyphenylalanine (DOPA) after inhibition of DOPA decarboxylase provided a measure of apparent DA synthesis. Mild stress increased the synthesis and release of DA in striatum. DA synthesis and release also were enhanced by the intra-striatal infusion of N-methyl-D-aspartate (NMDA), an agonist at NMDA receptors, and kainic acid, an agonist at the DL-a-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA)/kainate site. Stress-induced increase in DAsynthesis was attenuated by co-infusion of 2-amino-5-phosphonovalerate (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), antagonists of NMDA and AMPA/kainate receptors, respectively. In contrast, intrastriatal APV, CNQX, or kynurenic acid (a non-selective ionotropic glutamate receptor antagonist) did not block the stress-induced increase in DArelease. Stress-induced increase in DA release was, however, blocked by administration of tetrodotoxin along the nigrostriatal DA projection. It also was attenuated when APV was infused into substantia nigra. Thus, glutamate may act via ionotropic receptors within striatum to regulate DA synthesis, whereas glutamate may influence DA release via an action on receptors in substantia nigra. However, our method for monitoring DA synthesis lowers extracellular DA and this may permit the appearance of an intra-striatal glutamatergic influence by reducing a local inhibitory influence of DA. If so, under conditions of low extracellular DA glutamate may influence DA release, as well as DA synthesis, by an intrastriatal action. Such conditions might occur during prolonged severe stress and/or DA neuron degeneration. These results may have implications for the impact of glutamate antagonists on the ability of patients with Parkinson's disease to tolerate stress.

Keywords: Dopamine release; Dopamine synthesis; Excitatory amino acids Microdialysis; Parkinson's disease; Substantia nigra

Direct and indirect presynaptic control of dopamine release by excitatory amino acids by Dr. A. Chéramy; M. L'hirondel; G. Godeheu; F. Artaud; J. Glowinski (pp. 63-68).

Dopamine (DA) release from nerve terminals of the nigrostriatal DA neurons not only depends on the activity of nigral DA cells but also on presynaptic regulation. Glutamatergie neurons of cortical origin play a prominent role in these presynaptic regulations. The direct glutamatergic presynaptic control of DA release is mediated by N-methyl-D-aspartate (NMDA) andα-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) receptors, located on DA nerve terminals. In addition, by acting on striatal target cells, these glutamatergic neurons contribute also to indirect regulations of DA release involving several transmitters such as GABA, acetylcholine and neuropeptides. Diffusible messengers such as nitric oxide (NO) or arachidonic acid (AA) which are particularly formed under the stimulation of NMDA receptors may also participate to the regulation of DA release. In the present study, it will be shown that the co-application of NMDA and carbachol synergistically increases the release of [³H]-DA and that this effect is reduced by mepacrine or 4-bromophenacylbromide (10⁷M), two inhibitors of PLA2. Therefore endogenously released AA induced by the co-stimulation of NMDA and cholinergic receptors seems to be involved, at least partly, in the release of DA.

Keywords: Excitatory amino acids; Dopamine release; Mouse striatal microdiscs; Presynaptic mechanisms; Diffusible messengers; Arachidonic acid

Role of glutamate in neurodegeneration of dopamine neurons in several animal models of parkinsonism by Dr. P. K. Sonsalla; D. S. Albers; G. D. Zeevalk (pp. 69-74).

Although controversial, studies with methamphetamine and MPTP suggest a link between glutamate-mediated excitotoxicity and degeneration of dopamine cells. Both compounds are thonght to create a metabolic stress. To further explore glutamate actions in DA degeneration, we investigated the effects of other metabolic inhibitors. In mesencephalic cultures, DA cell loss produced by 3-NPA or malonate was potentiated by NMDA and prevented by MK-801. In vivo, striatal DA loss produced by intranigral infusions of malonate was also potentiated by intranigral NMDA and prevented by systemic MK-801. In contrast, systemic MK-801 did not prevent DA loss produced by intrastriatal malonate. Intrastriatal MK-801 or CGS 19755 did attenuate DA loss in METH-treated mice, but was confounded by the findings that METH-induced hyperthermia, an important component in toxicity, was also attenuated. Taken together, the data support the hypothesis of NMDA receptor involvement in degeneration of DA neurons. Furthermore, the data also suggest that this interaction is likely to occur in the substantia nigra rather than in the striatum.

Keywords: Glutamate; NMDA receptors; Dopamine; Malonate; Methamphetamine; MPTP; MK-801; Mesencephalic cultures; Mice; Basal ganglia

Blockade of glutamatergic transmission as treatment for dyskinesias and motor fluctuations in Parkinson's disease by L. Verhagen Metman MD; P. Del Dotto; P. J. Blanchet; P. van den Munckhof; T. N. Chase (pp. 75-82).

In animal models of Parkinson's disease (PD), glutamate antagonists diminish levodopa (LD)-associated motor fluctuations and dyskinesias. We sought to investigate if these preclinical observations can be extended to the human disease, by evaluating the effects of three non-competitive NMDA antagonists (dextrorphan, dextromethorphan and amantadine) on the motor response to LD in patients with advanced PD. In four separate trials, adjuvant therapy with these drugs reduced LD-induced dyskinesias and motor fluctuations. These findings support the view that drugs acting to inhibit glutamatergic transmission at the NMDA receptor can ameliorate LD associated motor response complications.

Keywords: NMDA antagonists; Motor response complications

Neurobiology of the nitric oxide in the nervous system by H. S. Sharma (pp. 83-85).

Nitric oxide as a peripheral and central mediator in temperature regulation by Prof. E. Simon M.D. (pp. 87-93).

In animals including humans nitric oxide (NO) serves as a biological messenger both peripherally at neuroeffector junctions and in the central nervous system where it modulates neuronal activity. Evidence for the involvement of NO in homeostatic control is accumulating also for temperature regulation in homeotherms. In the periphery an auxiliary role in the vasomotor control of convective heat transfer to heat dissipating surfaces and modulation of thermoregulatory heat generation, especially in brown adipose tissue as the site of nonshivering thermogenesis, are discussed as NO actions. At the central level a thermolytic role of NO in thermoregulation as well as in fever is assumed, however, experimental data opposing this view suggest that topical specificity may be important. At the level of single neurons, the observed interrelationships between thermosensitivity and responsiveness to NO are still not consistent enough to reconcile these data with the effects of NO-donors and inhibitors of NO-synthase on temperature regulation.

Keywords: Nitric oxide; Temperature regulation; Fever; Body temperature; Thermoregulatory effectors; Central neurons; Nitric oxide synthase; Cyclic GMP

Nitric oxide in the pathophysiology of hyperthermic brain injury. Influence of a new anti-oxidant compound H-290/51 by P. Alm; H. S. Sharma; S. Hedlund; P. -O. Sjöquist; J. Westman (pp. 95-103).

The possibility that nitric oxide (NO) is involved in the pathophysiology of brain injury caused by heat stress (HS) was examined using neuronal nitric oxide synthase (NOS) immunohistochemistry in a rat model. In addition, to find out a role of oxidative stress in NOS upregulation and cell injury, the effect of a new antioxidant compound H-290/51 (Astra Hässle, Mälndal, Sweden) was examined in this model. Subjection of conscious young rats to 4 h HS in a biological oxygen demand (BOD) incubator at 38°C resulted in a marked upregulation of NOS in many brain regions compared to control rats kept at room temperature (21 ± 1°C. This NOS immunoreactivity was found mainly in distorted neurons located in the edematous regions not normally showing NOS activity. Breakdown of the blood-brain barrier (BBB) permeability, increase in brain water content and marked neuronal, glial and myelin reaction were common findings in several brain regions exhibiting upregulation of NOS activity. Pretreatment with H-290/51 significantly attenuated the upregulation of NOS in rats subjected to HS. In these animals breakdown of the BBB permeability, edema and cell changes were considerably reduced. Our results suggest that hyperthermic brain injury is associated with a marked upregulation of NOS activity in the CNS and this upregulation of NOS and concomitant cell injury can be reduced by prior treatment with an antioxidant compound H 290/51. These observations indicate that oxidative stress seems to be an important endogenous signals for NOS upregulation and cell reaction in hyperthermic brain injury.

Keywords: Heat stress; Nitric oxide; Cell injury; Antioxidant; H-290/51Blood-brain barrier; Brain edema

Spinal nerve lesion induces upregulation of neuronal nitric oxide synthase in the spinal cord by T. Gordh; H. S. Sharma; P. Alma; J. Westman (pp. 105-112).

The possibility nitric oxide (NO) is involved the neurodegenrative mechanisms in the spinal cord following a chronic peripheral nerve lesion was examined using NOS immunohistochemistry. Spinal nerve lesion at L-5 and L-6 level was produced according to the Chung model, a model of neuropathic pain and rats were allowed to survive for 8 weeks. In one group of animals L-NAME was given intraperitoneally (1-2 mg/kg, i.p. daily) for 6 weeks. Sham operated rats, in which the spinal nerve was exposed but not ligated, served as controls. Ligation of spinal nerves in rats resulted in an upregulation of NOS which was most pronounced in the ipsilateral gray matter of the spinal cord compared to the contralateral side. In these rats, ultrastructural investigations showed distorted neurons, membrane disruption and myelin vesiculation. Sham operated rats did not show either NOS upregulation or structural changes in the spinal cord. Pretreatment with L-NAME significantly reduced NOS upregulation and the structural changes in the spinal cord were less pronounced. These observations strongly indicate a putative role of NOS in the pathophysiology of chronic nerve lesion. Our results may provide a new strategy to treat chronic neuropathic pain or to minimise neurodegeneration in the patients suffering from such diseases of the nervous system.

Keywords: Neuropathic pain; Spinal cord; Nitric oxide synthase; Neurodegeneration; L-NAME

Effect of glutamate and angiotensin II on whole cell currents and release of nitric oxide in the rat subfornical organ by Dr. H. A. Schmid (pp. 113-119).

Blood-borne angiotensin II (AngII) is known to mediate water-intake by its excitatory effect on neurons in the subfornical organ (SFO). Conversely, nitric oxide (NO) has exclusively inhibitory effects on rat SFO-neurons and on SFO-mediated water-intake. Extracellular and patch-clamp recordings from freshly dissociated rat SFO-neurons showed that glutamate activates Angll-sensitive SFO-neurons by opening ligand-gated cation channels. An immunocytochemical study showed that activation of glutamate receptors increased the concentration of the inhibitory second messenger cGMP in the SFO. A model is proposed suggesting that NO protects SFO-neurons from overexcitability by excitatory neurotransmitters.

Keywords: Angiotensin II; Patch clamp; Glutamate; NMDA; Nitric oxide; Thirst

Brain derived neurotrophic factor and insulin like growth factor-1 attenuate upregulation of nitric oxide synthase and cell injury following trauma to the spinal cord by H. S. Sharma Ph.D.; F. Nyberg; J. Westman; P. Alm; T. Gordh; D. Lindholm (pp. 121-129).

The possibility that brain derived neurotrophic factor (BDNF) and insulin like growth factor-1 (IGF) induced neuroprotectivn is influenced by mechanisms involving nitric oxide was examined in a rat model of focal spinal cord injury. BDNF or IGF-I (0.1 μg/10 [1 in phosphate buffer saline) was applied topically 30 min before injury on the exposed spinal cord followed by repeated doses of growth factors immediately before and 30 min after injury. Thereafter application of BDNF or IGF was carried out at every 1 h interval until sacrifice. Five hours after injury, the tissue pieces from the T9 segment were processed for nNOS immunostaining, edema and cell injury. Untreated injured rats showed a profound upregulation of nNOS which was most pronounced in the nerve cells of the ipsilateral side. A marked increase in the blood-spinal cord barrier (BSCB) permeability to¹²⁵I-albumin, water content and cell injury in these perifocal segments was also found. Pretreatment with BDNF and IGF significantly reduced the upregulation of nNOS in the spinal cord. This effect of the growth factors was most pronounced in the contralateral side. Rats treated with these neurotrophic factors showed much less signs of BSCB damage, edema and cell injury. These results suggest that BDNF and IGF pretreatment is neuroprotective in spinal cord injury and that these neurotrophic factors have the capacity to down regulate nNOS expression following trauma to the spinal cord. Our data provide new experimental evidences which suggest that BDNF and IGF may exert their potential neuroprotective effects probably via regulation of NOS activity.

Keywords: Brain derived neurotrophic factor; Insulin like growth factor-1; Nitric oxide; Spinal cord injury; Edema; Cell injury; Blood-spinal cord barrier; Immunohistochemistry

Spinal cord evoked potentials and edema in the pathophysiology of rat spinal cord injury by T. Winkler; H. S. Sharma Ph.D.; E. Stålberg; R. D. Badgaiyan; P. Alm; J. Westman (pp. 131-139).

The possibility that nitric oxide is somehow involved in the early bioelectrical disturbances following spinal cord injury in relation to the later pathophysiology of the spinal cord was examined in a rat model of spinal cord trauma. A focal trauma to the rat spinal cord was produced by an incision of the right dorsal horn of the T 10–11 segments under urethane anaesthesia. The spinal cord evoked potentials (SCEP) were recorded using epidural electrodes placed over the T9 and T12 segments of the cord following supramaximal stimulation of the right tibial and sural nerves in the hind leg. Trauma to the spinal cord significantly attenuated the SCEP amplitude (about 60%) immediately after injury which persisted up to 1h. However, a significant increase in SCEP latency was seen at the end of 5h after trauma. These spinal cord segments exhibited profound upregulation of neuronal nitric oxide synthase (NOS) immunoreactivity, and the development of edema and cell injury. Pretreatment with a serotonin synthesis inhibitor drug p-chlorophenylalanine (p-CPA) or an anxiolytic drug diazepam significantly attenuated the decrease in SCEP amplitude, upregulation of NOS, edema and cell injury. On the other hand, no significant reduction in SCEP amplitude, NOS immunolabelling, edema or cell changes were seen after injury in rats pretreated with L-NAME. These observations suggest that nitric oxide is somehow involved in the early disturbances of SCEP and contribute to the later pathophysiology of spinal cord injury.

Keywords: Nitric oxide; Spinal cord evoked potentials; Edema; Cell changes; p-CPA; Diazepam; Immunohistochemistry

Voltage-dependent sodium channels and excitatory amino acids by A. J. Carter (pp. 141-142).

Glutamate release inhibitors: A critical assessment of their action mechanism by Dr. T. P. Obrenovitch; J. Urenjak (pp. 143-150).

A number of important experimental data do not support the widespread hypothesis that Na⁺-channels block is cerebroprotective, essentially because it reduces presynaptic glutamate release: (i) the inhibition of exocytosis by these compounds is not specific to glutamate; (ii) aspartate efflux produced by various stimuli was also reduced, but aspartate cannot be released by exocytosis because it is not concentrated within presynaptic vesicles; and (iii) glutamate accumulated extracellularly during ischaemic or traumatic insult to the CNS is mainly of cytosolic origin. As an alternative, we propose that use-dependent Na⁺-channel blockers enhance the resistance of nerve cells to insults, primarily by decreasing their energy demand, and that reduced efflux of glutamate and other compounds is aconsequence of attenuated cellular stress.

Keywords: Glutamate release; Riluzole; Lamotrigine; Exocytosis; Excitotoxicity; Neuroprotection

Neuroprotection — rationale for pharmacological modulation of Na⁺-channels by Dr. J. Urenjak; T. P. Obrenovitch (pp. 151-158).

The primary factor detrimental to neurons in neurological disorders associated with deficient oxygen supply or mitochondrial dysfunction is insufficient ATP production relative to their requirement. As a large part of the energy consumed by brain cells is used for maintenance of the Na⁺ gradient across the cellular membrane, reduction of energy demand by down-modulation of voltage-gated Na⁺-channels is a rational strategy for neuroprotection. In addition, preservation of the inward Na⁺ gradient may be beneficial because it is an essential driving force for vital ion exchanges and transport mechanisms such as Ca²⁺ homeostasis and neurotransmitter uptake.

Keywords: Neuroprotection; Na⁺-channels; Na⁺-channel blockers; Ischaemia; Energy deprivation; Traumatic brain injury

The importance of voltage-dependent sodium channels in cerebral ischaemia by A. J. Carter (pp. 159-169).

Strategies for the treatment of thromboembolic stroke are based on restoring the blood flow as soon as possible and protecting the neurons from the deleterious consequences of cerebral ischaemia. Interest has focused on blockers of voltage-dependent Na⁺ channels as potential neuroprotective agents because they prevent neuronal death in various experimental models of cerebral ischaemia and act cytoprotectively in models of white matter damage. Although several Na⁺ blockers are currently being tested in various phases of clinical development, most of these agents are relatively weak and unspecific. I therefore consider it worthwhile to search for molecules which specifically block voltage-dependent Na⁺ channels for the treatment of cerebral ischaemia.

Keywords: Neuroprotection; Reperfusion; Na^- Channels; NMDA Receptors; Ischaemia; Thromboembolic stroke

Observations relative to the neurotoxicity and neurotoxic potential of amino acids by R. M. Kostrzewa (pp. 171-173).

Destruction of catecholamine-containing neurons by 6-hydroxydopa, an endogenous amine oxidase cofactor by Dr. R. M. Kostrzewa; R. Brus (pp. 175-179).

The amino acid, 6-hydroxydopa (6-OHDOPA), found at the active site of amine oxidases, exists as a keto-enol. Exogenously administered 6-OHDOPA is an excitotoxin likeβ-N-oxalylamino-L-alanine (BOAA) andβ-N-methylamino-L-alanine (BMAA), acting at the non-N-methyl-D-aspartate (non-NMDA)α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor. BMAA and BOAA are causal factors of neurolathyrism in humans. Much exogenously administered 6-OHDOPA is biotransformed by aminoacid decarboxylase (AADC) to the highly potent and catecholamine (CA) selective neurotoxin, 6-hydroxydopamine (6-OHDA). 6-OHDOPA destroys locus coeruleus noradrenergic perikarya and produces associated denervation of brain by norepinephrine-(NE) containing fibers. Opiopeptides and opioids enhance neurotoxic effects of 6-OHDOPA on noradrenergic nerves, by a naloxone-reversible process. An understanding of mechanisms underlying neurotoxic effects of 6-OHDOPA can be helpful in defining actions of known and newfound amino acids and for investigating their potential neurotoxic properties.

Keywords: 6-Hydroxydopa; 6-Hydroxydopamine; Noradrenergic neurons; Neurotoxicity; Excitatory amino acids

Molecular mechanisms of cellular injury produced by neurotoxic amino acids that generate reactive oxygen species by Dr. D. Metodiewa (pp. 181-187).

There is now strong experimental evidence that the basic precursors for the synthesis of catechol(amine) and indolamine neurotransmitters, tyrosine and tryptophan can act as generators of ROS (reactive oxygen species): peroxides, superoxide and peroxyradicals. The consequences of free radicals formation from precursors during oxidative degradation process, their possible participation in electron transfer/addition reactions and chain processes involving cell antioxidant defense system were presented and discussed. Although the generation of neurotoxic ROS by tyrosine and tryptophan is accepted to occur in the presented model systems, doubts can exist as to the situationin vivo, which may be completely different and remain to be explored. The relevance of the present findings with regard to a variety of neurological diseases cannot be ignored.

Keywords: Tyrosine; Tryptophan; Oxidative degradation; Chain processes; Scavenging; Cellular injuries analysis

Pharmacological approaches to counter the toxicity of dopa by .Dr. P. F. Von Voigtlander; G. J. Fici; J. S. Althaus (pp. 189-196).

Dopa and related catecholamines and their degradation products have been demonstrated to have neurotoxic potential in a number of cellular andin vivo experiments. Several mechanisms have been hypothesized to be involved including generation of prooxidant products that subsequently oxidize membrane lipids and exposed macromolecules. We have utilized a neuronal culture of cerebellar granule cells to study the toxicity of Dopa and the ability of various neuroprotective and antiparkinsonian compounds to offer protection therefrom. This model is apparently based on the ability of Dopa to non-enzymatically induce an oxidative injury to the neuronal cultures. Evidence for this arises from the equal neurotoxic potency of L- and D-Dopa in these cells and the ability of catalase, superoxide dismutase and glutathione to protect the neurons from this toxicity. Further, we found that the neuroprotective antioxidant, PNU-101033 is more effective and potent than vitamin E and deprenyl in this regard. Similarly the D2/D3 agonist, pramipexole is also capable of blocking Dopa toxicity in this model and this effect is independent of dopamine receptor affinity as both enantiomers are equally potent in this assay but disparate in receptor affinity. Also the protection by pramipexole is accompanied by the preservation of reduced glutathione. Thus, this activity seems to be a function of the oxidation potential of pramipexole and it's consequent antioxidant property. Potent antioxidants are effective blockers of Dopa toxicity. If the mechanisms involved in this toxicity have relevance to the progression of Parkinson's pathology in Dopa treated (or untreated) patients, these compounds have the potential to alter the course of the illness.

Keywords: Parkinson's disease; Neurotoxicity; Antioxidant; Pyrroloprimidine; Pramipexole; Vitamin E; Deprenyl

Release of endogenous excitatory amino acids in the neostriatum of the rat under physiological and pharmacologically-induced conditions by .Dr. M. Herrera-Marschitz; M. Goiny; Z. -B. You; J. J. Meana; E. Engidaworkl; Y. Chen; R. Rodriguez-Puertas; C. Broberger; K. Andersson; L. Terenius; T. Höpkfelt; U. Ungerstedt (pp. 197-203).

There is immunohistochemical evidence suggesting that glutamate (Glu) is released from nerve terminals and acts, via several receptor subtypes, as a major excitatory neurotransmitter in the cortico-striatal pathway of the rat. Aspartate (Asp) is also present in cortico-striatal neurons, but its role as a neurotransmitter has been questioned, since, in contrast to Glu, it has not been demonstrated in presynaptic vesicles. Glu and Asp can be found at subμM concentrations in the extracellular compartment of most areas of the basal ganglia. Their concentrations are largely regulated by transport mechanisms, but also by a synaptotagmin-dependent exocytotic release, and are sufficiently high to occupy junctional and extrajunctional receptors.We have investigated whether Glu and Asp release in the neostriatum can be selectively modulated by different neuronal systems. Dopamine (DA) and cholecystokinin (CCK) selectively stimulate Asp release, via D₁ and CCK_B receptor subtypes, respectively. Also opioid κ-agonists increase Asp release. We propose that the selective modulation of Asp release by D₁₋, CCK_B- and κ agonists involves striatal neurons containing Asp, but not Glu. In contrast, local perfusion with the ,μ-opioid antagonist D-Phe-Cys-Tyr-D-Trp-Orn-ThrPen-Thr-NH₂ (CTOP) increases both Glu and Asp release. This effect is probably exerted on cortico-striatal terminals, via presynaptic inhibitory μ-receptors. Thus, these results demonstrate that extracellular levels of Glu and Asp are modulated differentially by different neuronal systems, and suggest that in the neostriatum of the rat there are neuronal populations using Glu and/or Asp as messenger(s).

Keywords: Basal ganglia; Excitatory amino acids; Monoamines; Neuropeptides; Microdialysis; Immunocytochemistry; Parkinson's disease; Rat

Glycine_B recognition site of NMDA receptors and its antagonists by W. Danysz; C. G. Parsons (pp. 205-206).

Modulation of NMDA receptors by glycine — introduction to some basic aspects and recent developments by Dr. C. G. Parsons; W. Danysz; M. Hesselink; S. Hartmann; B. Lorenz; C. Wollenburg; G. Quack (pp. 207-216).

Glycine is a co-agonist at NMDA receptors and it's presence is a prerequisite for channel activation by glutamate or NMDA. Physiological concentrations reduce one form of NMDA receptor-desensitization. Interactions between the glycine_B site and other domains of the NMDA receptor are complex and include the glutamate, Mg⁺ and polyamines sites. Glycine shows different affinities at various NMDA receptor subtypes probably via to allosteric interactions between NMDA2 subunits and the glycine recognition site on the NMDAR1 subunit. There is still some debate whether the glycine_B site is saturatedin vivo but it seems likely that this depends on regional differences in receptor subtype expression, local glycine or D-serene concentrations and the expression of specific glycine transporters.Glycine_B antagonists and partial agonists have been reported to have good therapeutic indices as neuroprotective agents against focal ischaemia and trauma, anti-epileptics, anxiolytics, anti-psychotomimetics and in models of chronic pain. They clearly lack two potentially serious side effects classically associated with NMDA receptor blockade, namely neurodegenerative changes in the cingulate/retrosplenial cortex and psychotomimetic-like effects. This improved therapeutic profile may be partially due to the ability of full glycine_B antagonists to reveal Gycne-sensitive desensitization and possibly also via functional and/or regional NMDA receptor subtype selectivity.

Keywords: NMDA receptors; Glycine site; Antagonists; Partial agonists; Desensitisation; Subtypes

Effects of NMDA receptor antagonists on nociceptive responsesin vivo: Comparison of antagonists acting at the glycine site with uncompetitive antagonists by M. McClean; B. A. Chizh; P. M. Headley (pp. 217-221).

We have shown that members of a new series of tricyclic pyridophthalazine diones, defined as glycine_B site NMDA antagonistsin vitro, are selective and systemically active NMDA antagonistsin vivo. In electrophysiological tests in α-chloralose anaesthetised rats, these compounds reduced nociceptive reflex responses. In conscious rats they displayed analgesic properties. These glycine_B antagonists were compared electrophysiologically with several uncompetitive NMDA channel blockers. The degree of voltage dependence previously reportedin vitro related to the effectiveness of the agents against different amplitude nociceptive responses of spinal cord neuronesin vivo.

Keywords: Analgesia; NMDA; Electrophysiology; Behaviour; Voltage dependence

Novel glycine_B antagonists show neuroprotective activityin vivo by G. L. Wenk Ph.D.; L. M. Baker; B. Hauss-Wegrzyniakl; W. Danysz; J. D. Stoehr (pp. 223-226).

The degeneration or dysfunction of cholinergic neurons within the basal forebrain of patients with Alzheimer's disease (AD) may be related to the vulnerability of these cells to endogenous glutamate (Beal, 1995; Greenamyre and Young, 1989). The administration of drugs that attenuate the toxic actions of glutamate in the early stages of the disease might significantly delay its rate of progression. Two approaches to neuroprotection from endogenous glutamatergic function were investigated and found to be effective: blockade of voltage-dependent, NMDA-type glutamate receptor channels and antagonism of an NMDA-receptor related glycine_B modulatory site.

Keywords: Glycine; Basal forebrain; Neuroprotection; Neurotoxicity; Acetylcholine

Ligands of the NMDA receptor-associated glycine recognition site and motor behavior by B. D. Kretschmer (pp. 227-234).

Motor behavior critically depends on glutamatergic functions in the basal ganglia (BG). The dorsal and ventral striatum — the main input structures of the BG - are involved in modulation of stereotyped sniffing behavior, locomotion, catalepsy and prepulse inhibition. The effects of the NMDA receptor have been well characterized in respect to motor behavior in the past. The function of the allosteric glycine site was however disregarded until now, because brain penetrating ligands were missing. The present study summarized the motor behavioral profile of several glycine site ligands (7chlorokynurenate, ACEA 1021, MRZ-2/576, (+) HA-966, D-cycloserine and felbamate). It is shown that through blockade of the glycine site of the NMDA receptor a distinct behavioral profile can be obtained.

Keywords: Glycine site; Locomotion; Stereotypy; Catalepsy; NMDA; Motor behavior

Glycine_B antagonists as potential therapeutic agents by Dr. W. Danysz; C. G. Parsons; M. Karcz-Kubichal; A. Schwaier; P. Popik; K. Wedzony; J. Lazarewicz; G. Quack (pp. 235-239).

It is not clear what therapeutic application is most likely for agents blocking glycine site of the NMDA receptors (glycine_B). Majority of the studies to date used either glycine_B antagonists with doubtful brain penetration or partial agonists. Following systemic administration to rats of our newly developed glycme_B antagonists (MRZ 2/570; 2/571 and 2/576) and L-701,324 (MSD) as a reference agent the following behavioural effects were observed: weak (if any) antiparkinsonian-like effects, lack of anxiolytic activity, inhibition of physical and motivational aspects of morphine dependence and neuroprotective activity in global ischaemia. The side effects include: sedation, ataxia, and myorelaxation. We detected neither vacuolisation in the cingulate cortex nor impairment of pre-pulse inhibition indicating lack of psychotomimetic potential.

Keywords: NMDA receptors; Glutamate; Behaviour; MRZ 2/570; MRZ 2/576; L-701,324

Neuroactive tryptophan metabolites: focus on kynurenines by J. Luthman (pp. 241-241).

Regulation of kynurenic acid levels in the developing rat brain by R. Schwarcz Ph.D.; B. Poeggeler; A. Rassoulpour; G. Ceresoli-Borroni; P. S. Hodgkins (pp. 243-249).

Several brain-specific mechanisms control the formation of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) in the adult rat brain. Two of these, dopaminergic neurotransmission and cellular energy metabolism, were examined in the brain of immature (postnatal day 7) rats. The results indicate that during the early postnatal period cerebral KYNA synthesis is exceptionally amenable to modulation by dopaminergic mechanisms but rather insensitive to fluctuations in cellular energy status. These findings may be of relevance for the role of KYNA in the function and dysfunction of the developing brain.

Keywords: Excitotoxicity; Glia; Kynurenines; Neuroprotection; Ontogeny

Expression of the kynurenine enzymes in macrophages and microglial cells: regulation by immune modulators by D. Alberati-Giani; A. M. Cesura (pp. 251-255).

The regulation of the expression of indoleamine 2,3-dioxygenase (IDO) was studied in cloned murine macrophages (MT2) and microglial (N11) cells. Both cell lines express IDO and inducible nitric oxide synthase activity after interferon-γ (IFN-γ) stimulation. The regulation of IDO expression appears to differ in the two cell lines. Nitric oxide (NO) production negatively modulates the expression of IDO activity in IFN-γ-primed macrophages, thereby indicating a cross-talk between the kynurenine and nitridergic pathways in these cells. Conversely, this down-regulation of IDO activity by NO does not occour in microglial cells. A differential regulation of IDO expression in the two cell lines was also observed with LPS and picolinic acid. Together with previous findings, these results indicate the existence of marked differences in the regulation of the expression of the kynurenine pathway enzymes between macrophages and microglial cells.

Keywords: Macrophages/monocytes; Microglia; Cytokines; Indoleamine 2,3-dioxygenase; Nitric oxide synthase; Nitric oxide

Role of zinc in blockade of excitotoxic action of quinolinic acid by picolinic acid by Dr. K. H. Jhamandas; R. J. Boegman; R. J. Beninger; S. Flesher (pp. 257-261).

This study examined whether picolinic acid (PIC) inhibits quinolinic acid (QUIN) — induced excitotoxicity through zinc chelation. Injection of QUIN into the nucleus basalis magnocellularis significantly depleted cortical choline acetyltransferase activity 7 days post injection and PIC inhibited this response. Zinc augmented the QUIN- but not NMDA-induced response. When PIC was co-administered with zinc, PIC failed to attenuate the QUIN-induced response. The inhibition of QUIN — induced cholinergic toxicity by PIC may involve chelation of zinc.

Keywords: Picolinic acid; Quinolinic acid; Zinc; Excitotoxicity

Effects of the 3-hydroxyanthranilic acid analogue NCR-631 on anoxia-, IL-1β- and LPS-induced hippocampal pyramidal cell lossin vitro by J. Luthman; A. -C. Radesäter; C. Öberg (pp. 263-269).

The kynurenine pathway intermediate 3-hydroxyanthranilic acid (3-HANA) is converted by 3-HANA 3,4-dioxygenase (3-HAO) to the putative neuropathogen quinolinic acid (QUIN). In the present study, the neuroprotective effects of the 3-HANA analogue and 3-HAO inhibitor NCR-631 was investigated using organotypic cultures of rat hippocampus. An anoxic lesion was induced by exposing the cultures to 100% N₂ for 150 min, resulting in a pronounced loss of pyramidal neurons, as identified using NMDA-R1 receptor subunit immunohistochemistry. NCR-631 provided a concentration-dependent protective effect against the anoxia. NCR-631 was also found to counteract the loss of pyramidal neurons in two models of neuroinflammatory-related damage; incubation with either LPS (10 ng/ml) or IL-1β (10 IU/ml). The findings suggest that NCR-631 has neuroprotective properties and that it may be a useful tool to study the role of kynurenines in neurodegeneration.

Keywords: Kynurenine pathway; 3-Hydroxyanthranilic acid; Neurodegeneration; Anoxia; Neuroinflammation; Interleukin-1

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.14, #1-3)