Increased neuronal nitric oxide synthase expression in brain following portacaval anastomosis

It has previously been suggested that increases of l-arginine uptake into brain following portacaval shunting may result in increased activities of constitutive neuronal nitric oxide synthase (nNOS). In order to further address this issue, nNOS protein and gene expression were studied by Western blot analysis using a monoclonal nNOS antibody and RT-PCR respectively in the brains of rats following portacaval shunting or sham operation. Portacaval shunting resulted in a 2-fold increase (P<0.01) in nNOS protein and a concomitant 2.4-fold increase (P<0.01) in nNOS mRNA. Increased nNOS activity in brain and the resulting increase in nitric oxide production could contribute to the increased cerebral blood flow and to the pathogenesis of hepatic encephalopathy in chronic liver disease.     

Behavioral symptoms in adult rats after postnatal l-nitro-arginine

We addressed experimentally the suggestion by Gally et al. [Gaily J. A., Read Montague P., Reeke G. N. Jr and Edelman G. M. (1990) Proc. Natl Acad. Sci. U.S.A.87, 3547–3551] that nitric oxide may play a role in the use-dependent modification of synaptic efficacy in the developing nervous system. In a preliminary control experiment, we treated rat pups from postnatal day 8 to postnatal day 22 with a nitric oxide synthase blocker (l-nitro-arginine) and compared their growth curves and brain weights to those of saline injected control pubs. No significant differences were found after the 14 days of nitric oxide synthase inhibition. In the subsequent experiment, we inhibited nitric oxide synthesis in rat pups from postnatal day 8 to day 29 and assessed their place learning ability and open field behavior as adults. We found an increased speed of habituation of locomotion in an open field in 5-month-old rats that had been treated postnatally with a nitric oxide synthase blocker. There was no difference between treated and nontreated rats with respect to place learning in a water maze. We conclude that perturbation of nitric oxide production during early postnatal development does not preclude normal learning and memory function in the adult.     

Modulation of extracellular glutamate concentration by nitric oxide synthase inhibitor in rat transient forebrain ischemia

The purpose of the present study was to clarify the effect of topical administration of a nitric oxide synthase inhibitor on extracellular glutamate concentration in transient forebrain ischemia. Two microdialysis probes were inserted into the bilateral striata of Wistar rats. N^G-Nitro-l-arginine (l-NNA) with or withoutl-arginine was topically administered into the unilateral striatum through one of the microdialysis probes, while Ringer's solution was perfused into the contralateral striatum as the control, and 14 minutes of forebrain ischemia was applied. The extracellular glutamate concentration during ischemia and subsequent reperfusion was statistically significantly higher on the 100 μMl-NNA-perfused side than on the control side, but 1 MMl-NNA was ineffective. When 100 μMl-NNA was perfused together with 500 μMl-arginine, the glutamate concentration did not differ from that on the control side. Moreover, administration of 500 μMl-arginine significantly suppressed the glutamate elevation after reperfusion. The fact that the lower dose ofl-NNA increased the accumulation of glutamate during ischemia and reperfusion without altering the blood flow may indicate that nitric oxide affords protection against ischemic neuronal damage. However, since the higher dose ofl-NNA did not affect the glutamate concentration, it appears that the effect of nitric oxide on extracellular glutamate concentration in forebrain ischemia differs, depending on the degree of the inhibition of NOS activity.     

N-methyl-d-aspartate stimulates dopamine release through nitric oxide formation in the nucleus accumbens of rats

Intracerebral microdialysis technique was utilized to study the effect ofN^G-nitro-l-arginine, a nitric oxide (NO) synthase inhibitor, onN-methyl-d-aspartate (NMDA)-induced dopamine overflow in the nucleus accumbens of unanesthetized, freely moving rats. Perfusion of 1 and 3 mM NMDA through the microdialysis probe dose-dependently increased the extracellular dopamine level in the nucleus accumbens. Coapplication of 0.5 mMd-(−)-2-amino-5-phosphonovaleric acid (D-AP5), a selective and competitive NMDA receptor antagonist, significantly reduced the dopamine overflow induced by 3 mM NMDA. Perfusion of 0.5 mMN^G-nitro-l-arginine alone did not affect the basal dopamine level, whereas it suppressed the NMDA-evoked dopamine overflow in the nucleus accumbens when concurrently applied with 3 mM NMDA. These results suggest that NO mediates, at least in part, dopamine release resulting from NMDA receptor activation in the nucleus accumbens of rats.     

Higher free D-aspartate and N-methyl-D-aspartate levels prevent striatal depotentiation and anticipate L-DOPA-induced dyskinesia

In Parkinson's disease (PD) progressive alteration of striatal N-methyl-d-aspartate receptors (NMDARs) signaling has emerged as a considerable factor for the onset of the adverse motor effects of long-term levodopa (l-DOPA) treatment. In this regard, the NMDAR channel blocker amantadine is so far the only drug available for clinical use that attenuates l-DOPA-induced dyskinesia (LID). In this study, we examined the influence of a basal corticostriatal hyper-glutamatergic transmission in the appearance of dyskinesia, using a genetic mouse model lacking d-Aspartate Oxidase (DDO) enzyme (Ddo^−/− mice). We found that, in Ddo^−/− mice, non-physiological, high levels of the endogenous free d-amino acids d-aspartate (d-Asp) and NMDA, known to stimulate NMDAR transmission, resulted in the loss of corticostriatal synaptic depotentiation and precocious expression of LID. Interestingly, the block of depotentiation precedes any change in dopaminergic transmission associated to 6-OHDA lesion and l-DOPA treatment. Indeed, lesioned mutant mice display physiological l-DOPA-dependent enhancement of striatal D1 receptor/PKA/protein phosphatase-1 and ERK signaling. Moreover, in line with synaptic rearrangements of NMDAR subunits occurring in dyskinetic animal models, a short l-DOPA treatment produces a dramatic and selective reduction of the NR2B subunit in the striatal post-synaptic fraction of Ddo^−/− lesioned mutants but not in controls. These data indicate that a preexisting hyper-glutamatergic tone at NMDARs in Ddo^−/− mice produce abnormal striatal synaptic changes that, in turn, facilitate the onset of LID.     

Different mechanisms of l-arginine induced dilation of brain arterioles in normotensive and hypertensive rats

We evaluated the response of pial arterioles to l-arginine in anesthetized normotensive rats and spontaneously hypertensive rats equipped with a closed cranial window. Topical application of 10⁻⁶ − 10⁻⁴ mol/l l-arginine, which is known to be the endogenous substrate for the synthesis of nitric oxide, induced dose-dependent arteriolar vasodilation. The response was more pronounced in hypertensive than in normotensive rats (at the concentration of 10⁻⁴ mol/l l-arginine: 18.3 ± 3.3% vs. 6.7 ± 1.7%, respectively, means ± S.E.). The stereoisomer d-arginine had no effect in hypertensive rats. Topical application of the nitric oxide synthase inhibitor N-nitro-l-arginine converted l-arginine-induced dilation to constriction in normotensive and hypertensive rats. The cyclooxygenase inhibitor indomethacin (5 μg/ml cerebrospinal fluid) also blocked the dilation in both strains. Photochemical endothelial injury blocked l-arginine-induced dilation in normotensive rats, but only partly antagonized the response in hypertensive animals. Intravenous or topical pretreatment with the free radical scavenger superoxide dismutase significantly reduced the dilating response to 10⁻⁴ mol/l l-arginine in hypertensive rats. Superoxide dismutase did not significantly change the response to l-arginine in normotensive animals. It is concluded that nitric oxid formation in the endothelium and liberation of cyclooxygenase products cause l-arginine-induced dilation in normotensive rats. While nitric oxide and cyclooxygenase products are also involved in l-arginine-induced dilation in spontaneously hypertensive rats, superoxide radicals contribute to the enhanced response in this strain. This mechanism appears to be specific for the hypertensive animals and is only partly dependent on an intact endothelium.     

Effects of Central Injection of Kyotorphin and l-Arginine on Oxytocin and Vasopressin Release and Blood Pressure in Conscious Rats

Intracerebroventricular (ICV) administration of an inhibitor of nitric oxide synthase (NOS) increases oxytocin but not vasopressin secretion, in dehydrated rats [38]. Surprisingly, central injection of l-arginine, the substrate for NOS, caused a similar effect. Kyotorphin (l-tyrosyl-l-arginine), a dipeptide formed from l-arginine by kyotorphin synthetase in the brain may mediate this magnocellular response. Therefore, the dose and time responses of hormone release were compared following ICV injection of kyotorphin and l-arginine to conscious rats that were normally hydrated or deprived of water for 24 h. In water-sated rats, both l-arginine and kyotorphin increased blood pressure and plasma glucose levels coincident with elevating circulating levels of oxytocin, but not vasopressin. In dehydrated animals, both l-arginine and kyotorphin increased plasma oxytocin levels with a similar time course but only kyotorphin decreased vasopressin release. d-arginine, like l-arginine, stimulated secretion of oxytocin, indicating a nonstereospecific effect. A kyotorphin receptor antagonist (l-leucyl-l-arginine) given ICV to dehydrated animals elevated plasma oxytocin and prevented the decrease in vasopressin levels after kyotorphin. Thus, kyotorphin, but not l-arginine, appears to attenuate release of vasopressin either directly from magnocellular neurons or indirectly via modulating compensatory reflexes activated by the pressor response. On the other hand, an excess of l-arginine and kyotorphin within the CNS may mimic the stress response by augmenting release of oxytocin and activating the sympathetic nervous system to increase blood pressure and plasma glucose levels.     

Nitric oxide reduces body temperature and sympathetic input to brown adipose tissue during PGE1-hyperthermia

The firing rate of the nerves innervating interscapular brown adipose tissue (IBAT) and IBAT and colonic temperatures (T_IBAT and T_c) were monitored in urethane-anaesthetized male Sprague-Dawley rats. These variables were measured for 40 min before (baseline values) and 40 min after a 4 μmoles l-arginine (l-arg) or 400 nmoles nitroprusside (NP) injection in a lateral cerebral ventricle and an intracerebroventricular administration of 500 ng prostaglandin E₁ (PGE₁). The same variables were monitored in other rats with l-arg or NP or PGE₁ administration alone. No drug was injected in control rats. The results show that l-arg or NP injection reduces the increases in firing rate, T_IBAT, T_c induced by PGE₁ These findings suggest that nitric oxide is important in the control of thermogenic changes during the PGE₁ hyperthermia.     

GABAB receptors modulate synaptically-evoked responses in the rat dentate gyrus, in vivo

We assessed the effects of systemically injected baclofen, a GABA_B agonist, on single and paired-pulse responses in the dentate gyrus of urethane-anesthetized rats, in vivo. Baclofen (10 mg/kg) significantly increased the duration of single excitatory responses. This increase was blocked by the GABA_B receptor antagonist, CGP 35348, indicating that baclofen was acting through GABA_B receptors. To determine the mechanism underlying this increase in response duration, the NMDA antagonist,d-2-amino-5-phosphonopentanoic acid (d-APV), was administered intracerebroventricularly (i.c.v.) after baclofen.d-APV by itself had no effect on the duration of the population excitatory post-synaptic potential (EPSP). However, when infused after baclofen,d-APV blocked the baclofen induced increase in EPSP duration. This indicates the prolonged EPSP duration caused by baclofen resulted from an enhancement of an NMDA receptor mediated component of the response. We then examined the effect of baclofen on population responses to paired stimuli. Baclofen attenuated paired-pulse inhibition of population spike amplitudes at a 25 ms interstimulus interval. CGP-35348 reduced the effect of baclofen on paired-pulse inhibition, indicating that baclofen suppressed paired-pulse inhibition by acting on GABA_B receptors. In contrast to its disinhibitory effect at the 25 ms interval, baclofen had an inhibitory effect on responses evoked at a 150 ms interstimulus interval. Under control conditions, we observed that when stimuli were delivered 150 ms apart, both the EPSP duration and population spike amplitude evoked by the second stimulus were enhanced. Baclofen suppressed this enhancement. We conclude that GABA_B receptor activation by baclofen reduces short interval inhibition and this reduction of inhibition leads to an enhanced NMDA-receptor mediated response. We also conclude that by reducing inhibition, baclofen occludes paired-pulse disinhibition found at longer interstimulus intervals. In contrast, CGP-35348 directly blocks paired-pulse disinhibition. Thus, GABA_B receptor activation can regulate synaptic inhibition, in turn influencing NMDA receptor activation and synaptic transmission. Thus, pharmacologic manipulation of GABA_B receptors may provide a powerful approach for regulating synaptic transmission in the hippocampus.     

Chronic administration of l-NAME in drinking water alters working memory in rats

To examine the role of nitric oxide (NO) in the maintenance of working memory of rats, the effects of chronic administration (in drinking water) of the NO synthase inhibitor, N^w-nitro-l-arginine methyl ester (l-NAME), on this behavior was examined with a simple test of remembering recently explored objects. Unlike other working memory tasks that require a subject to perform for a reward such as food or water or to avoid shock, our task measured spontaneous exploration of novel and familiar objects and has been described as a “pure” working memory task [9]. Normal subjects spend significantly more time in contact with new environmental components and less time with familiar objects. A subject that extensively reexplores a stimulus with which it has previous experience is presumed to exhibit some memory loss associated with the object. Memory changes were evaluated by measuring the relative time subjects explored familiar versus new stimulus objects. Rats (n = 15) that chronically drank l-NAME (≈ 90 mg/kg/day) for 14 days spent significantly less time exploring a novel object than did rats (n = 13) that drank only tap water (p < .05). This effect of t.-NAME was abolished by concurrent administration of l-Arginine (≈ 4.5 g/kg/day). Total object exploration was not affected by our drug treatments, suggesting that our object discrimination task is not activity dependent. These data are consistent with the hypothesis that NO is required for some forms of working memory.     

l-NAME inhibits pentylenetetrazole and strychnine-induced seizures in mice

Nitric oxide (NO) formation has been shown in many neuronal tissues subserves a variety of functions. N-Methyl-d-aspartate (NMDA) receptor stimulation which releases nitric oxide and raises cGMP levels, mediates epileptiform activity induced by various agents. Disinhibition of inhibitory neurotransmitter γ-aminobutyric acid (GABA) and/or activation of NMDA receptor appears to be factors involved in the initiation and generalization of the pentylenetetrazole (PTZ) induced seizures. In the present study, we examined the effects of N^ω-nitro-l-arginine methylester (l-NAME) which inhibits nitric oxide synthase, on PTZ and strychnine induced seizures in mice. l-NAME (100 mg/kg) significantly prolonged the onset time of tonic generalized extension without affecting myoclonic jerks and tonic-clonic convulsions. l-NAME (200 mg/kg) significantly delayed three characteristic behavioral changes including first myoclonic jerk (FMJ), generalized clonic seizure (GCS) and tonic generalized extension (TGE). The effects of l-NAME were reversed by l-arginine (1000 mg/kg). l-NAME (100 and 200 mg/kg) significantly delayed the onset time of strychnine induced TGE. The effects of both doses of l-NAME were reversed by l-arginine. In conclusion, our results demonstrate that NO synthase inhibition suppresses the onset time of PTZ and strychnine induced seizures. Under the light of our current knowledge NO synthase inhibitors seem far away to be considered as a group of antiepileptic drugs. On the other hand there are some strong evidences about the role of NO in central pathophysiological mechanisms. © 1997 Elsevier Science B.V. All rights reserved.     

β-Endorphin Inhibition of Endogenous Norepinephrine Release From the A2 Noradrenergic Nucleus In Vitro: Role of Mu Opiate Receptors and Na Ion Permeability

An in vitro approach was used to determine the opioid receptor subtype mediating β-endorphin inhibition of endogenous norepinephrine release from the A2 nucleus in the caudal dorsomedial medulla of rats. The voltage-sensitive Na⁺ channel blocker tetrodotoxin was used to investigate the role of Na⁺-dependent action potentials in β-endorphin inhibition of K⁺-evoked norepinephrine release. Human β-endorphin_1–31 inhibited K⁺-evoked norepinephrine release in a concentration-dependent fashion. Activation of δ- and χ-opioid receptors had no effect on endogenous norepinephrine release. The inhibitory effect of β-endorphin was blocked in a concentration-dependent manner by the μ-opioid receptor antagonist CTOP (Cys², Tyr³, Orn⁵, Pen⁷ amide). Tetrodotoxin (TTX) inhibited norepinephrine release evoked by 25 mM K⁺ in a concentration-dependent manner and blocked the inhibitory effects of β-endorphin. These results indicate that β-endorphin acts on μ-opioid receptors to inhibit K⁺-evoked norepinephrine release from A2 neurons and suggest that the receptors involved are not located on noradrenergic nerve terminals.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Distribution of glutamate receptor subunits in the primate temporal cortex and hippocampus

The distribution of subunits for the N-methyl-d-aspartate (NR1, NR2A/B), α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (GluR1, GluR2/3, GluR4) and low affinity kainate (GluR5/6/7) ionotropic glutamate receptors was examined by immunocytochemistry in the temporal cortex and hippocampus of the rhesus macaque (Macaca mulatta). Neurons expressing NR1, NR2A/B, GluR2/3, and GluR4 subunits were widely distributed in all of the cortical layers but the overall density of the GluR4-immunopositive neurons was very low. Neurons expressing the GluR1 subunit were found predominantly in cortical layers V and VI while those expressing the GluR5/6/7 subunits were concentrated in layer V and were readily distinguishable by the thick elongate shape of their primary apical dendrites. Subcellular differences in the immunostaining pattern were also noted between the different glutamate receptor subunits. NR1 and NR2A/B immunoreactivity was most pronounced in somatic and primary dendritic compartments and to a lesser extent in cortical and hippocampal molecular layers. GluR1 immunoreactivity was more intense than GluR2/3 in the hippocampal molecular layers whereas GluR4 was undetectable. GluR5/6/7 immunoreactivity was very intense in the dentate molecular layer, and the CA1 pyramidal cells had a subcellular distribution of GluR5/6/7 that was similar to the cortical neurons. Overall, the distribution patterns of the different glutamate receptor subunits was identical in animals that had been ovariectomized and in ovariectomized animals that had subsequently undergone estradiol or estradiol/progesterone hormone replacement. Taken together, these findings demonstrate a differential spatial arrangement of glutamate receptor subunits in the primate temporal cortex and hippocampus, which may have functional significance for the integration of excitatory inputs to these areas. Furthermore, they show that in adult macaques, sex steroids do not play a major role in determining the distribution patterns of these receptor subunits.     

Dietary goitrogen-induced changes in the transport of 2-deoxy-d-glucose and amino acids across the rat blood-brain barrier

The hypothesis that a defect in the rate-limiting blood-brain barrier (BBB) nutrient transport may be one of the factors responsible for the brain defects seen in some iodine deficiency disorders was tested in Wistar/NIN rats fed potassium thiocyanate (KSCN), a synthetic goitrogen. The BBB nutrient transport was measured by the brain uptake index (BUI) method. Feeding KSCN to female rats (from weaning) through their growth, pregnancy and lactation (G1) but not from conception (G2) or parturition (G3) resulted in a significant decrease (≈23%) in the BBB transport of 2-deoxy-d-glucose (2-DG) in their offspring at weaning, compared with controls (C). Post-weaning KSCN-feeding (G4) of control pups did not affect their BBB 2-DG transport (BUI: 36.2±4.98, vs 38.8±4.11). The effects of different KSCN regimens on BBB transport of leucine (leu), tyrosine (tyr) and sucrose (background marker) were inconsistent, of smaller magnitude (≈10%) and appeared to be of little significance. Withdrawing KSCN from the diet of chronically KSCN-fed (G1) mothers from conception (G5) or parturition (G6) prevented the impairment of BBB 2-DG transport in pups (BUI: 27.0±4.98, 20.8±3.27, 26.9±3.99 and 28.3±3.47 in C, G1, G5 and G6 pups, respectively); this was reversed by feeding the control diet to G1 pups from weaning. Withdrawal of dietary KSCN did not affect BBB transport of leu, tyr and sucrose. The decreased BBB transport of 2-DG in G1 pups appears to be due to a decrease in affinity (K_t app 5.46 vs 4.15 mM) rather than in the capacity (T_max app 0.94 vs 0.91 μmoles/g/min) of the transport system. Intracarotid injections of KSCN per se had no effect on BBB 2-DG transport, suggesting that the effects may be secondary to the altered thyroid status of the animal.     

Differential effects of NMDA and non-NMDA antagonists on the activity of aromatic l-amino acid decarboxylase activity in the nigrostriatal dopamine pathway of the rat

This study examined the acute effects of a variety of NMDA and non-NMDA antagonists on the activity of aromatic l-amino acid decarboxylase (AADC) in the corpus striatum (CS) and substantia nigra (SN) of the rat. Sixty min pretreatment with the high affinity NMDA receptor-channel blockers MK 801 (0.01, 0.1 and 1 mg/kg) and phencyclidine (4 mg/kg) elevated AADC activity in both the CS and SN (2- to 3-fold). Even more striking increases in AADC were noted with 40 mg/kg amantadine (3.8-fold for CS, 9.0-fold for SN), 40 mg/kg memantine (3.4-fold for CS, 3.1-fold for SN; 20 mg/kg no effect) and 40 mg/kg dextromethorphan (3.4-fold for CS, 6.2-fold for SN, in 6/10 `responders'). Similarly pronounced increases in AADC activity in CS (1.9-fold) and SN (2.8-fold) were detected after administering clonidine (2 mg/kg). R-HA 966 (5 mg/kg, not 1 mg/kg) modestly raised AADC activity in CS (0.45-fold) and not SN. Other drugs had no effect on the activity of the decarboxylase enzyme, including CGP 40116 (1 and 5 mg/g), eliprodil (10 mg/kg), NBQX (10 mg/kg, 30 min pretreatment) and atropine (1 mg/kg). These experiments indicate that blocking the NMDA receptor-channel (and to a lesser extent the glycine site) or stimulating α₂-adrenoceptors, profoundly increases AADC activity, more especially in the SN than CS. By contrast, inhibiting the NMDA glutamate recognition or polyamine sites, AMPA or muscarinic receptors is without effect on AADC in either brain region. The ability of amantadine and memantine to potentiate the antiparkinsonian actions of l-DOPA in the clinic, may be due to facilitated decarboxylation of l-DOPA by the brain.     

Neurotoxicity, Blood-Brain Barrier Breakdown, Demyelination and Remyelination Associated With NMDA-Induced Lesions of the Rat Lateral Hypothalamus

Excitotoxins have been widely used to make lesions in the brains of experimental animals because they have the ability to destroy neurones while sparing fibres of passage. Because loss of fibres of passage can confound the interpretation of lesion effects, this property is of considerable value. Recently, however, there have been reports indicating that excitotoxins acting at different sites within the rat CNS not only destroy neurones but also strip myelin from fibres and compromise the integrity of the blood-brain barrier. However, some reports also indicate that the myelin content of the lesioned area recovers. Excitotoxic lesions of the lateral hypothalamus have been shown to produce local demyelination. The present studies sought to investigate this effect further by (1) defining the time course of demyelination and possible remyelination after excitotoxic lesions of the lateral hypothalamus made with n-methyl-d-aspartate (NMDA); (2) establishing the relationships between neuronal loss, de- and remyelination after various doses of NMDA; and (3) examining the integrity of the blood-brain barrier using an immunohistochemical probe. Our data show that after injection of NMDA into the lateral hypothalamus there was neuronal loss, blood-brain barrier disruption (followed by recovery over approximately 12 days), triggering of reactive gliosis, invasion of the lesioned area by cells from outwith the CNS, demyelination over an area coexistent with but not exceeding the area of neuronal loss, and remyelination. Remyelination occurred over a period of 3 months following the production of the lesion and was associated initially with blood vessels. It occurred across the whole of the lesioned area, not by encroachment from the borders. All doses of NMDA that produced neuronal death also produced demyelination. These data confirm that excitotoxic lesions of the lateral hypothalamus demyelinate fibres, but show for the first time that remyelination occurs here. They are consistent with reports concerning excitotoxin actions at other CNS sites and indicate that de- and remyelination after excitotoxic lesions is a ubiquitous process. Consideration should be given to this when using excitotoxins to make fibre-sparing lesions.     

Correlation between the Movement Disorders Society Unified Parkinson's Disease rating scale (MDS-UPDRS) and the Unified Parkinson's Disease rating scale (UPDRS) during L-dopa acute challenge

While Movement Disorders Society Unified Parkinson’s Disease rating scale (MDS-UPDRS) validation has been exhaustive; performance evaluation to detect acute changes arising after administration of a single dose of l-dopa has yet to be explored. To determine the correlation between UPDRS and MDS-UPDRS during the acute challenge with ldopa and the MDS-UPDRS equivalent to 30% cutoff score of UPDRS for defining responsiveness, 64 patients were assessed. Consecutive assessments were performed immediately before and after administration of a single dose of l-dopa/carbidopa 250/25 mg using the motor section of the UPDRS and the MDS-UPDRS. Good diagnostic accuracy, consistent with published findings of high correlation between scales was observed. Area under the curve (AUC) was 0.99 (CI = 0.97–1.00, P < 0.001) and maximum Youden index (Y = 0.905) corresponded to a cutoff of 24.5%. In conclusion we have found an excellent correlation between UPDRS and MDS-UPDRS and that the 30% of variation in UPDRS score used for predicting sustained long term l-dopa response was equivalent to 24% in MDS-UPDRS.     

Regulation of cyclic GMP levels in the rat frontal cortex in vivo: effects of exogenous carbon monoxide and phosphodiesterase inhibition

A microdialysis method combined with a sensitive radioimmunoassay was used to monitor cGMP release in the frontal cortex of the anesthetized rats in vivo. We assessed the relative contribution of endogenous nitric oxide (NO), and effects of exogenous carbon monoxide (CO) and phosphodiesterase activity, as possible regulators of cortical CGMP levels. Perfusion with CO-saturated aCSF (≈1 mM CO) failed to significantly stimulate cortical cGMP levels. For comparison, cerebellar cGMP levels increased by 2-fold during CO stimulation, followed by a prolonged response that was fully reversible with the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME). Cortical perfusion with zinc protopophyrin-IX (100 μM), a widely used inhibitor of the CO-generating enzyme heme oxygenase, suppressed cGMP levels by 50%, a response that spontaneously recovered in spite of the continuous presence of the metalloporphyrin. Perfusion with isobutylmethyl xanthine IBMX (1 mM) resulted in 5-fold increase in cortical cGMP levels, as compared to basal levels without IBMX. In the presence of IBMX, l-NAME suppressed basal cortical cGMP levels by 70% indicating that NO synthase activity generates the bulk of cGMP in this brain region, as previously shown for basal cGMP production in the hippocampus and the cerebellum. These data also emphasize a crucial role for phosphodiesterase activity in the maintenance of cGMP levels in vivo in the frontal cortex. The relatively weak responses to exogenous CO lend little support for a role of this gas in regulating basal cortical cGMP levels in vivo.     

The role of nitric oxide in the inhibitory effect of ghrelin against penicillin-induced epileptiform activity in rat

Ghrelin, a gastric peptide with key action on food intake, has been recently recognized as a potential antiepileptic agent. In the present study, we investigated the involvement of nitric oxide in the effect of ghrelin on penicillin-induced epileptiform activity in rat. Thirty minutes after penicillin injection, ghrelin, at doses of 0.5, 1, 2 μg, was administered intracerebroventricularly (i.c.v.). Ghrelin, at a dose of 1 μg, significantly decreased the mean frequency of epileptiform activity without changing the amplitude whereas other doses of ghrelin (0.5 and 2 μg) did not alter either the mean of frequency or amplitude of epileptiform activity. The effects of systemic administration of nitric oxide synthase (NOS) inhibitors, non-selective N^G-nitro-l-arginine methyl ester (l-NAME), selective neuronal NOS inhibitor, 7-nitroindazole (7-NI) and NO substrate, l-arginine on the anticonvulsive effects of ghrelin were investigated. The administration of l-NAME (60 mg/kg, i.p.), 15 min before ghrelin (1 μg) application, reversed the anti-epileptiform effects of ghrelin whereas 7-NI (40 mg/kg, i.p.) did not influence it. The present study provides electrophysiological evidence that the intracerebroventricular injection of ghrelin has an inhibitory effect against epileptiform activity in the penicillin model of epilepsy. The anti-epileptiform activity of ghrelin was reversed by nonspecific nitric oxide synthase inhibitor l-NAME, but not selective neuronal nitric oxide synthase inhibitor 7-NI, indicating that ghrelin requires activation of endothelial-NOS/NO route in the brain.