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The present study shows that nicotinamide prevents the long-term effect of perinatal asphyxia on dopamine release monitored with in vivo microdialysis in the neostriatum of 3-month-old rats. Perinatal asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 16 or 20 min. Sibling, spontaneous, and caesarean-delivered pups were used as controls. Saline or nicotinamide (0.8 mmol/kg, i.p.) was administered to control and asphyxia-exposed animals 24, 48, and 72 h after birth. After weaning, the rats were randomly distributed in laboratory cages for animal care under standard ad libitum laboratory conditions. Approximately 3 months after birth, control and asphyxia-exposed animals were implanted with microdialysis probes into the lateral neostriatum for measuring extracellular monoamine and metabolite levels with HPLC-coupled to an electrochemical detection system under basal, D-amphetamine, and K(+)-depolarising conditions. There was an asphyxia-dependent decrease of extracellular dopamine levels, mainly observed during the periods when D-amphetamine (100 microM) or KCl (100 mM) was added into the perfusion medium. Compared to that observed in caesarean-delivered controls, the effect of D-amphetamine on dopamine levels was decreased by approximately 30 and 70% in animals exposed to 16 and 20 min of perinatal asphyxia, respectively. The effect of K(+)-depolarisation was decreased by 45 and 83% in animals exposed to the same periods of asphyxia, respectively. Both effects were prevented by nicotinamide, even if the treatment started 24 h after the insult. The present results support the idea of nicotinamide as an interesting molecule, useful for protecting against anoxia/ischemia occurring at neonatal stages. Nicotinamide can help to restore NADH/NAD+ depletion, but also to inhibit PARP-1 overactivation, a mechanism of action that has attracted attention, representing a novel target for neuroprotection following insults involving energy failure.

Elevated levels of glutamate, an endogenous excitatory amino acid, contribute to the development of neuronal injury in various cerebral diseases. Using a microdialysis approach, the response of extracellular levels of amino acids and metabolic parameters to glutamine synthetase inhibition by l-methionine sulfoximine was monitored simultaneously in the hippocampal formation and in the frontal cortex of the rabbit brain. In the hippocampal formation the decrease of glutamine levels during l-methionine sulfoximine treatment was more pronounced than in the frontal cortex, and was accompanied by a delayed decline of extracellular glutamate concentrations. Furthermore, l-methionine sulfoximine diminished the increase of lactate and pyruvate concentrations in the hippocampal formation, but not in the frontal cortex. Neither l-methionine sulfoximine treatment nor microdialysis probe insertion caused neuronal apoptosis, as measured by in situ tailing. An impaired function of hippocampal astrocyte glutamate uptake mechanisms or a higher functional capacity of the cortical glutamine synthetase may be possible explanations for the differences demonstrated. The present data are in accordance with regional differences in glutamine synthetase activation during bacterial meningitis and may explain, in part, the higher susceptibility of certain areas of the hippocampal formation (i.e., the dentate gyrus) to neuronal injury.

Asphyxia during birth can cause gross brain damage, but also subtle perturbations expressed as biochemical or motor deficits with late onset in life. Thus, it has been shown that brain dopamine levels can be increased or decreased depending upon the severity of the insult, and the region where the levels are determined. In this study, perinatal asphyxia was evoked by immersing pup-containing uterus horns removed by hysterectomy in a water bath at 37 degrees C for various periods of time from 0 to 20 min. After the insult, the pups were delivered, given to surrogate mothers, treated with nicotinamide, further observed and finally, 4 weeks later, killed for monoamine biochemistry of tissue samples taken from substantia nigra, neostriatum and nucleus accumbens. The main effect of perinatal asphyxia was a decrease in dopamine and metabolite levels in nucleus accumbens, and a paradoxical increase in the substantia nigra. Nicotinamide (100 mg/kg i.p., once a day for 3 days, beginning 24 h after the perinatal asphyctic insult) prevented the effect of asphyxia in nucleus accumbens. Furthermore, striatal dopamine levels were increased by nicotinamide in asphyctic animals. No apparent changes were observed in substantia nigra. A prominent unexpected effect of perinatal asphyxia alone was on the levels of the metabolite of 5-hydroxytryptamine, 5-hydroxyindoleacetic acid (5-HIAA), which were increased in substantia nigra and decreased in both neostriatum and accumbens. However, nicotinamide increased 5-HIAA levels in all regions, which appeared to be related to the extent of the asphyctic insult. These results suggest that nicotinamide is a useful treatment against the long-term consequences produced by perinatal asphyxia on brain monoamine systems, and that there is a therapeutic window following the insult, providing a therapeutic opportunity to protect the brain.

This study investigated the influence of temperature or glutamate antagonism on the immediate outcome of perinatal asphyxia. Perinatal asphyxia was produced by water immersion of fetus-containing uterus horns removed by cesarean section from ready to deliver rats. The uterus horns were kept in a water bath for different time periods, before the pups were delivered and stimulated to breathe. After delivery, the pups were assessed for behavior and for systemic glutamate, aspartate, lactate and pyruvate levels measured with in vivo microdialysis, or ex vivo for energy-rich phosphates, including adenosine triphosphate (ATP), in brain, heart and kidney. In a series of experiments, asphyxia was initiated in a water bath at 37 degrees C, before the pup-containing uterus horns were moved for different time intervals to a 15 degrees C bath. In another series of experiments, the mothers were treated with N-methyl-D-aspartate (NMDA) antagonist, dizocilpine (MK-801), or alpha-amino-3-hydroxy-methylisoxazole-4-propionic acid (AMPA) antagonist,2,3-dihydroxy-6-nitro-7-sulfamoyl benzo(f) quinoxalin NBQX) 1 h before hysterectomy and asphyxia at 37 degrees C. The rate of survival rapidly decreased following exposure to more than 16 min of asphyxia, and no survival could be observed after 22 min of asphyxia. An LD50 was estimated to occur at approximately 19 min of asphyxia. The outcome was paralleled by a decrease in ATP in kidney, followed by a decrease in heart and brain. A maximal decrease in ATP was observed after 20 min of asphyxia in all tissues. Systemic microdialysis revealed that glutamate, aspartate and pyruvate levels were increased with a peak after 5 min of asphyxia. In contrast, lactate levels increased along with the length of the insult. Survival was increased when the pup-containing uterus horns were moved from a 37 degrees C to a 15 degrees C bath, at 15 min of asphyxia (the LD50 was thus increased to 30 min). If the shift occurred at 10 or 5 min of asphyxia, the LD50 increased to 80 or 110 min, respectively. The effect of glutamate antagonism was minor compared to hypothermia; the best effect (an increase in the LD50 to approximately 22 min) was observed after combining AMPA and NMDA antagonists.

Aspartate and glutamate were monitored in the scala tympani of the guinea pig cochlea using in vivo microdialysis before and during noise exposure. Moderate level broad band noise [105 dB sound pressure level (SPL), 30 min] neither altered the levels of aspartate or glutamate, nor auditory brainstem response (ABR) thresholds. High level noise exposure (135 dB SPL, 30 min) caused a large increase in aspartate (330%), a smaller increase in glutamate (150%), and a permanent ABR threshold shift of 60-75 dB between 2.0 and 12.5 kHz. Morphological analysis of the cochlea revealed a collapse of supporting structures, swelling of the afferent dendrites under the inner hair cells, and outer hair cell loss. Pretreatment with the NMDA antagonist, MK 801 (1 mg/kg body weight, i.p.) 1 h before noise exposure protected the afferent dendrites from swelling but did not protect the collapse of supporting structures, outer hair cell loss, or auditory thresholds. In conclusion, the noise-induced increase in aspartate and glutamate release in the cochlea and the protective effect of NMDA antagonism suggest that these two neurotransmitters are involved in noise-induced hearing loss.

We have used the perfused guinea-pig temporal-bone preparation to study the sound-evoked efflux of aspartate and glutamate, which are putative afferent transmitters in the cochlea. The cochlea was stimulated with white noise at 89, 95, and 101 dB SPL. Cochlear function was monitored by recording the endocochlear potential, the cochlear microphonic, and the summating potential. In silence, there was a low basal efflux of both amino acids. A significant and intensity dependent sound-evoked efflux of aspartate was observed at all levels, whereas a significant efflux of glutamate was found only at the 101 dB SPL level. Immunohistochemistry of sections from the organ of corti showed an ubiquitous distribution of glutamate-like immunoreactivity in the sensory organ and ganglion, whereas aspartate-like immunoreactivity was found in the region of the inner hair cells and in the spiral ganglion. In view of these findings, we suggest that not only glutamate, but also aspartate may have a neurotransmitter role in the afferent pathway of the cochlea.

The effect of sulphated cholecystokinin-8 (CCK-8S) on extracellular dynorphin B, aspartate, glutamate and GABA levels in the rat fronto-parietal cortex was investigated with in vivo microdialysis. The peptide was infused through the microdialysis probe trying to mimic local CCK-8S release. Basal levels of dynorphin B were around 20 pM, aspartate 100 nM, glutamate 600 nM and GABA 30 nM. CCK-8S (10 microM) induced a approximately 3-fold increase in extracellular dynorphin B, aspartate and glutamate levels, while GABA levels were only slightly increased. The effect of CCK-8S was restricted to the stimulated neocortex. Systemic pretreatment with the CCKB antagonist, L-365, 260, but not with the CCKA antagonist, L-364, 718, significantly antagonised the effect of CCK-8S on cortical dynorphin B and aspartate release. However, both CCKA and CCKB antagonists inhibited the increase in cortical glutamate levels. Thus, the present results indicate that cortical CCK release exerts a stimulatory modulation on cortical dynorphin B and aspartate release via the CCKB receptor subtype, and on glutamate release via both CCKA and CCKB receptor subtypes. Considering electrophysiological evidence that CCK increases neuronal firing rates in many brain regions, it may be suggested that CCK represents a stimulatory system modulating the function of the neocortex.

In vivo microdialysis was used to study the effect of secretogranin II-derived peptides on dynorphin B (Dyn B), dopamine, gamma-aminobutyric acid (GABA), glutamate and aspartate release in the substantia nigra and neostriatum of halothane-anaesthesized rats. In the substantia nigra, local infusion of secretoneurin (secretogranin II 154-186) (1-50 microM) increased, in a concentration-dependent manner, extracellular aspartate, glutamate, Dyn B, dopamine and GABA levels. The effect was particularly prominent on aspartate and glutamate levels which, following 50 microM of secretoneurin, were increased by > 20 and > 10 fold, respectively. However, the effect of secretoneurin on Dyn B release appeared to be more specific, since a significant increase (> 20 fold) was already observed following 1 microM of secretoneurin. In the neostriatum, Dyn B, glutamate, aspartate and GABA levels were also increased by local secretoneurin infusion, but the effect was less prominent than in the substantia nigra. In the substantia nigra, only Dyn B levels were significantly increased following infusion of 10 microM of the secretoneurin-C terminal (secretoneurin-15C), whereas Dyn B and GABA levels were increased by the same concentration of the secretogranin II C terminus (YM). Only glutamate and aspartate levels were increased by local infusion of 10 microM of secretogranin II 133-151 (LF), a peptide adjacent to secretoneurin in the primary amino acid sequence. In the neostriatum, Dyn B and GABA levels were increased by 10 microM of secretoneurin-15C. Dyn B levels were also increased by 10 microM of YM, and glutamate and aspartate levels were increased by 10 microM of both YM and LF. Thus secretogranin II-derived peptides affect extracellular levels of several putative neurotransmitter systems monitored in the basal ganglia of the rat with in vivo microdialysis. The effect of Dyn B appears to be specific and related to a physiological role of secretoneurin, since (i) it occurs in an area where secretoneurin-immunocytochemistry has been observed, (ii) is exerted at comparatively low concentrations, and (iii) is mimicked by secretoneurin-15C. The increases in excitatory amino acid levels produced by high concentrations of secretoneurin and other secretogranin II-derived peptides reflect, perhaps, a potential neurotoxicity produced by abnormal accumulation of these peptides.

We have studied the ability of the vigilance-promoting drug modafinil to counteract the ischemic lesion produced by a unilateral microinjection of endothelin-1 (ET-1) in the neostriatum of the rat using a combined morphometrical, biochemical, cardiovascular and behavioral analysis. ET-1 was injected unilaterally into the neostriatum. The ET-1-induced lesion volume, which was determined by a computer-assisted morphometrical analysis, was reduced by the 7-day modafinil treatment (10, 30, and 100 mg/kg i.p.) in a dose-related way. Modafinil also produced a dose-related counteraction of the ET-1-induced increase of perfusate lactate levels, as determined by intrastriatal microdialysis without affecting the ET-1 induced reduction of striatal blood flow, as determined by laser-Doppler flowmetry. The ipsilateral rotational behavior induced by apomorphine in the ET-1-lesioned rats was reduced dose-dependently by modafinil treatment. Thus, morphological, neurochemical, and behavioral evidence that the putative ischemic striatal injury induced by microinjection of ET-1 in the rat neostriatum is counteracted in a dose-dependent way by modafinil treatment has been obtained. The mechanism does not appear to involve an increase in striatal blood flow. It is instead speculated that its powerful preventive action in striatal ischemic injury may be related to a reduced anaerobic metabolism.

The present study examines the possibility that lesions induced by intrastriatal injections of endothelin-1 (ET-1, 0.43 nmol/0.5 microliter) are ischemic in nature due to a vasoconstriction of the cerebral microvessels. In time course and dose-response experiments with ET-1 and in comparisons with ET-3, the volume of the lesions has been determined based mainly on the disappearance of striatal nerve cells, using a computer assisted morphometrical analysis. The blood flow in the neostriatum close to the site of injection of ET-1 was determined acutely by Laser-Doppler flowmetry. The acute metabolic effects of ET-1 were also studied on striatal superfusate levels of lactate, pyruvate, dopamine and its metabolites DOPAC (3,4-dihydroxyphenylacetic acid) and homovanillic acid (HVA) using an intrastriatal microdialysis probe. Dose related striatal lesions were observed with ET-1 (0.043-0.43 nmol) with a peak lesion volume after 24-48 h and the possible existence of a penumbra area. ET-3 showed a reduced potency to produce striatal lesions compared to ET-1. The lesions induced by ET-1 were prevented by coinjection with dihydralazine, a vasodilator drug. Acutely ET-1 (0.43 nmol/0.5 microliter) produced a prolonged reduction of the cerebral blood flow down to 40% of control values and temporary increases of striatal lactate and DA efflux, the latter change being very marked. Also a significant reduction of DOPAC and HVA was observed. These neurochemical changes were all prevented by treatment with dihydralazine.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastrin levels, in the peripheral venous blood of conscious dogs treated with apomorphine (0.05 mg/kg IV), were analysed with a radioimmunoassay. Pretreatment (30 min) with the gastric acid inhibitors cimetidine, ranitidine (H2 receptor antagonists, 4 mg/kg and 1 mg/kg respectively) or omeprazole (H+-K+ ATPase inhibitor, 1.6 mg/kg) prolonged the elevation of gastrin levels occurring in response to an administration of apomorphine. Haloperidol (0.1 mg/kg), but not the peripheral dopamine receptor antagonist domperidone (0.2 mg/kg), abolished the enhanced gastrin response to apomorphine occurring after pretreatment with cimetidine. Cimetidine did not increase the gastrin response to apomorphine in vagotomized dogs. The results are interpreted in terms of an additive gastrin response to apomorphine (different from the short lasting initial peak previously described) which is vagally mediated and inhibited by the gastric acid.

Apomorphine (0.05 mg/kg intravenously) was given to conscious dogs, and gastrin levels were measured in peripheral venous blood with a radioimmunoassay. Apomorphine induced an increase of gastrin levels which peaked at 5 min. The peripheral dopamine D-2/DA2 receptor antagonist domperidone (0.2 mg/kg), but not halopemide (0.1-1 mg/kg) nor the D-1/DA1 receptor antagonist SCH 23,390 (0.1 mg/kg), blocked the gastrin response to apomorphine. Both domperidone and halopemide, but not SCH 23,390, blocked the apomorphine-induced vomiting. These results suggest that apomorphine increases gastrin levels by an action at D-2/DA2 receptors, which are situated outside the blood brain barrier and differ from the receptor inducing the vomiting.

The effect of neuroleptics on the hypothalamopituitary-adrenal system has been early recognized, but never adequately related to antipsychotic or side effects produced by dopamine antagonists. We are now presenting results showing that the newly characterized dopamine D-1 receptor antagonist, SCH 23390 (0.1 mg/kg i.v.) as well as the mainly dopamine D-2 receptor antagonists, haloperidol (0.1 mg/kg i.v.) and chlorpromazine (1 mg/kg i.v.), produced an increase of cortisol levels (108, 144 and 226% respectively, 20 min after the injection) determined by radioimmunoassay in blood samples collected from superficial veins of the legs of conscious dogs. The 5-HT2 receptor antagonist, cyproheptadine (0.2 mg/kg i.v.), did not modify the cortisol levels. These results suggest that cortisol increase is an effect common to neuroleptic compounds, independently of their relative antagonistic action at dopamine D-1 or D-2 receptors.