R. Lucchi

Neuronal nitric oxide synthase is permanently decreased in the cerebellum of rats subjected to chronic neonatal blockade of N-methyl-d-aspartate receptors

Pharmacological blockade of the (NMDA) receptor at critical stages of brain development may have long-lasting effects on brain chemistry and on animal behavior. We report here experiments in which the competitive NMDA receptor antagonist CGP 39551 was administered to rat pups from postnatal day 7 (P7) to P18. The stage of treatment was selected to primarily target the cerebellum, whose granule cells undergo post-mitotic migration and establishment of synaptic connections during this period. We focused our study on the long-term consequences of CGP 39551 treatment on the neuronal isoform of nitric oxide synthase (nNOS) since nNOS is highly expressed in the cerebellum and it is functionally linked to the NMDA receptor. Treated rats exhibited a long-lasting (up to P70) decrease in the intensity of nNOS immunocytochemical staining in the cerebellar cortex accompanied by a decrement of calcium-dependent NOS catalytic activity. A comparable decrease of enzyme activity was measured in the cerebral cortex, but not in the hippocampus, of adult rats. Other neurochemical markers (glutamatergic, gabaergic, purinergic) and glutamine synthetase were unchanged, while a cholinergic marker was slightly increased in the cerebellum of CGP 39551 treated animals. Taken together these data show that blockade of NMDA receptor during the critical period of formation and stabilization of neuronal circuits preferentially affects long-term nNOS expression and catalytic activity in the cerebellum.

Functional adenosine al receptors in goldfish brain: Regional distribution and inhibition of K+-evoked glutamate release from cerebellar slices

In goldfish brain, [3H]cyclohexyladenosine binding sites are ubiquitously distributed with a maximum in the hypothalamus and a minimum in the spinal cord. The binding parameters measured in cerebellar membranes (Kd = 0.88 +/- 0.08 nM; Bmax = 59.65 +/- 2.62 fmol/mg protein) are not significantly different from those of the whole brain. In perfused goldfish cerebellar slices, stimulation of cyclic AMP accumulation by 10(-5) M forskolin was markedly reduced (58.7%) by treatment with 10(-4) M cyclohexyladenosine, an adenosine A1 receptor agonist, and the reduction was reversed in the presence of 10(-4) M 8-cyclopentyltheophylline, a selective A1 receptor antagonist. In the same brain preparation, 30 mM K+ stimulated the release of glutamate, glutamine, glycine and GABA in a Ca(2+)-dependent manner, whereas the aspartate and taurine release was Ca(2+)-independent. Cyclohexyladenosine inhibited the 30 mM K(+)-evoked release of glutamate in a dose-related manner. This effect was reversed by 8-cyclopentyltheophylline. These results support the hypothesis that adenosine A1 receptors present in goldfish cerebellum are involved in the modulation of glutamate transmitter release.

Characterization of A1 adenosine receptors in membranes from whole goldfish brain

Abstract 1. 1. A1 adenosine receptor binding was investigated using the selective agonist [3H]-cyclohexl-adenosine ([3H]CHA) on membranes prepared from adult (12–14 cm length) goldfish whole brain. 2. 2. The A1 receptor agonist [3H]-cyclohexyladenosine bound saturably, reversibly and with high affinity (Kd = 1.20 ± 0.08 nM; Bmax = 6.8 ± 3.02 fmol/mg protein). 3. 3. The specific binding was increased by Mg2+ and inhibited by guanosine 5′-triphosphate (Ki = 5.81 ± 1.54 μM). 4. 4. In equilibrium competition experiments, the adenosine analogs R-phenylisopropyladenosine, cyclohexyladenosine, N-ethylcarboxamidoadenosine, 2-chloroadenosine, S-phenylisopropyladenosine and the antagonists 8-cyclopentyl-1,3-dipropylxanthine and theophylline all displaced [3H]-cyclohexyladenosine from high-affinity binding sites with the rank order of potency in displacing characteristics of an A1 adenosine receptor.

Neurochemical Changes in Cerebellum of Goldfish Exposed to Various Temperatures

Acclimation of goldfish at 35°C increased the cerebellar content of aspartate, glutamate, and taurine and [3H]glutamate uptake. Acclimation at 4°C increased the levels of glutamine, serine, and alanine and glutamine synthetase (GS) activity. Adenosine content increased in cerebellum of fish acclimated to warm temperature. K+-evoked release of endogenous and exogenous glutamate from cerebellar slices increased in fish acclimated at 35°C compared to 4°C. The basal level of cyclic adenosine 3′:5′-monophosphate (cAMP) in perfused cerebellar slices in fish acclimated at 35°C was much higher than in fish acclimated at 5° and 22°C. It is concluded that variations of environmental temperature produces large neurochemical changes in goldfish cerebellum.

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [3H]cyclohexyladenosine (CHA) to the cellular fractions and P2 subfractions of the goldfish brain was studied. The A1 receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P2), 30 mM K+ stimulated glutamate, taurine and GABA release in a Ca(2+)-dependent fashion, whereas the aspartate release was Ca(2+)-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 microM) inhibited K(+)-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K+ (30 mM) stimulated Ca2+ influx (46.8 +/- 6.8%) and this increase was completely abolished by pretreatment with 100 nM omega-conotoxin. Pretreatment with 100 microM R-PIA or 100 microM CHA, reduced the evoked increase of intra-synaptosomal Ca2+ concentration, respectively by 37.7 +/- 4.3% and 39.7 +/- 9.0%. A possible correlation between presynaptic A1 receptor inhibition of glutamate release and inhibition of calcium influx is discussed.

Spontaneous recovery of MPTP-damaged catecholamine systems in goldfish brain areas

In goldfish, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered for 3 consecutive days (10 mg/kg), produced a marked decrease in dopamine (DA) and noradrenaline (NA) levels in telencephalon, diencephalon and medulla oblongata, without affecting the serotonin (5-HT) content. Furthermore the neurotoxin decreased either [3H]DA high affinity uptake or K(+)-stimulated DA release from synaptosomal (P2) preparations, with concomitant up-regulation of D2 postsynaptic receptors as well. No significant changes of choline acetyltransferase and glutamic acid decarboxylase activity or [3H]glutamate uptake were observed. Moreover the pretreatment with deprenyl (1 mg/kg) or mazindol (10 mg/kg) but not with clorgyline (5 mg/kg) prevented catecholamine depletion. Added in vitro to synaptosomal preparations both MPTP and more potently MPP+, in a concentration-dependent manner, inhibited [3H]DA uptake. Time course study revealed that MPTP-induced alteration of neurochemical parameters in goldfish brain areas were almost completely reversed within 6 weeks, suggesting that catecholamine systems in goldfish brain show a remarkable power of recovery after MPTP lesion.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in goldfish cerebellum: neurochemical and immunocytochemical analysis

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered in goldfish for 3 consecutive days (10 mg kg-1 i.p.), caused cerebellar disappearance of dopamine-hydroxylase (DBH) immunoreactive fibres, whereas the noradrenergic cell bodies located in the medulla oblongata appeared intact. This effect was coupled with marked decreases in cerebellar noradrenaline (NA) and dopamine (DA) levels. An increase of immunostaining for glial fibrillary acidic protein (GFAP) was also observed. In the cerebellum of MPTP-treated fish, the contents of glutamate and GABA were significantly reduced, whereas glutamine was strongly increased. These modifications were concomitant with a significant increase of glutamine synthetase (GS) activity, whereas glutamic acid decarboxylase (GAD) activity was decreased. No changes in choline acetyltransferase (ChAT) and ornithine decarboxylase (ODC) activities were observed. High affinity uptake of glutamate and GABA was strongly reduced. Pretreatment of fish with either the monoamine oxidase inhibitor pargyline or the catecholamine (CA) uptake blocker mazindol largely prevented such modifications. The NMDA-sensitive glutamate receptor uncompetitive antagonist, dizocilpine maleate (MK-801), failed to protect against MPTP-induced damage. In conclusion, the neurotoxic effects of MPTP in goldfish cerebellum appear to be not specific against catecholaminergic terminals and could promote astrocytic reactions.

Reversible effects of 1-methyl-4-phenylpyridinium ion (MPP+) on dopaminergic neurons in goldfish retina

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridinium ion (MPP(+)) on the dopamine (DA), norepinephrine (NA) and serotonin (5-HT) levels in goldfish retina, was investigated. Intravitreal (i.v.) injection of MPTP (10 ?g) caused a 60-70% depletion of DA, which was prevented by the simultaneous administration of pargyline (10 ?g). A similar dopamine depletion was observed with MPP(+) (10 ?g). In contrast, NA or 5-HT levels were unaffected by MPTP or MPP(+). Time-course studies revealed that MPP(+)-induced DA depletion in goldfish retina was almost completely reversed within 2 months. The results confirm the selective action of MPP(+) towards dopaminergic neurons in goldfish retina.

Reversible changes in goldfish brain polyamine concentrations and synthetic enzymes after cold exposure.

Exposure of goldfish to the cold (5 degrees C) caused a sharp increase in brain putrescine level during the first week. Such increase continued at a minor rate for the whole period of exposure (2 months). In contrast, the content of spermidine and spermine remained unchanged. Putrescine increase was concomitant with a remarkable rise in ornithine decarboxylase activity (ODC), which reached a maximum stimulation after 1 week of cold exposure, and declined thereafter, remaining significantly higher than the control for the entire period of study. Cold exposure caused also a reduction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and an increase of ornithine level, whereas methionine content was unchanged. When fish exposed to cold temperature were returned to 20 degrees C, the modifications observed on brain polyamine metabolism were completely reversed. Supported by previous observations, our results suggest that the changes in the polyamine metabolism induced in goldfish brain by cold exposure could represent an homeostatic mechanism carried out by the goldfish to minimize the possible effects of thermal changes.

Effect of cold exposure on polyamine levels and ornithine decarboxylase activity of goldfish tissues

Goldfish exposed to the cold (5 °C) for a week exhibited modifications in polyamines metabolism, as shown by the increase of putrescine levels in liver, heart, muscle and brain, whereas the content of spermidine and spermine was unchanged. Putrescine increases in tissues considered were concomitant with a remarkable rise in ornithine decarboxylase activity. Kinetic analysis of brain enzyme activity showed that the apparent Km was unchanged by cold exposure, whereas Vmax was strongly increased.