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Clinical observations suggest that bilirubin encephalopathy is often seen in newborn infants presenting not only with hyperbilirubinemia but also with alterations in oxygen transport like in severe anaemia. Since bilirubin and hypoxia have been shown to be detrimental to the central nervous system, the present study was designed to test the additional effects of the two insults on the outcome of cultured neurons from the forebrain of 14 day-old embryos. After 6 days in vitro, neurons were exposed either to bilirubin (0.5 microM) or to bilirubin and hypoxia for 6 hours. Thereafter, cells were reoxygenated for 96 hours in standard conditions. Control cells were kept in normoxia. Cell viability was assessed by the methyltetrazolium method. Cell death (apoptosis or necrosis) was characterized by fluorescent nuclear staining with DAPI. Rates of protein synthesis and energy metabolism in neurons were measured by [3H]leucine and [3H]2-deoxyglucose incorporation, respectively. Data are reported as percentages of change as compared to controls. Each experiment involved 5 to 10 dishes per time point and was repeated 2 to 4 times.Bilirubin reduced cell viability by 24.5% vs controls (p0.001) at 96 h while 16% of the neurons exhibited morphological features of apoptosis (p0.001). The combination of hypoxia with bilirubin induced a 34% decrease in cell viability (p0.001) and the percentage of apoptotic cells was higher than after the exposure to hypoxia or to bilirubin alone. The rate of protein synthesis increased significantly in all experimental conditions as early as 1 h after the onset of the insult and at 48 h post reoxygenation. It increased again at 72 h in the cells exposed to bilirubin or to bilirubin and hypoxia. These sequential changes in synthesis of specific proteins seem to be involved in delayed neuronal death. Bilirubin decreased significantly [3H]2-deoxyglucose incorporation at 24 h while it increased when the neurons were exposed to both bilirubin and hypoxia (+60%, p0.001) and decreased thereafter. These data confirm the deleterious effects of bilirubin on neuronal viability, on protein synthesis and metabolic rates. The combination of bilirubin with hypoxia resulted in stronger detrimental effects on neurons than bilirubin alone.

Generalized seizures induced a widespread upregulation of adenosine A1 receptors linked to G proteins in the rat brain. Changes in receptor density were more pronounced in structures mediating seizure activity and were age-dependent.

The effects of feeding a diet enriched in caffeine or carbamazepine (CBZ) were investigated in rats in a quantitative autoradiographic study of adenosine A1 receptors (labeled by [3H]cyclohexyladenosine, [3H]CHA) and adenylate cyclase (labeled by [3H]forskolin). Although regional distribution of [3H]CHA and [3H]forskolin binding sites differed in some areas, chronic CBZ as well as chronic caffeine upregulated both of them. The changes in receptor densities occurred in the same brain microregions, suggesting that caffeine and CBZ act as antagonists at similar subpopulations of adenosine A1 receptors and [3H]forskolin binding sites. Therefore, a selective interaction of these two drugs with distinct adenosine A1 receptors (and adenylate cyclase) probably does not explain the differential effects of caffeine and CBZ on neuronal activity.

During the past years the number of biological roles proposed for adenosine and for precursor adenine nucleotides in the central nervous system has increased considerably. In addition to its role in intermediary metabolism, adenosine displays a number of receptor-mediated physiological actions which include dilatation of cerebral vesseis (Winn et al. 1981a). Adenosine also acts as a neuromodulator or neurotransmitter, inhibiting neuronal firing (Phillis et al. 1979) and synaptic transmission (Kuroda et al. 1978) as well as altering cyclic AMP concentrations in brain tissues (Sattin and Rail 1970; Daiy, 1977). In addition the relatively selective ability of methylxanthines such as caffeine and theophylline to block effects of adenosine on neuronal firing and cvciic AMP formation. suggest that pharmacological actions of these widely used substances may be mediated by blockade of central adenosine receptors (Snyder et al. 1981). Such receptors have been visualized by autoradiography on axon terminals of excitatory neurons (Goodman et al. 1983). Thus adenosine properties are those of local hormone which is released from the nerve cells. Indeed, a release of adenosine derivatives that Is accelerated by electrical stimulation has been observed from Isolated brain tissue (Pull and Mcilwain 1972a).

Nerve endings (synaptosomes) prepared from guinea-pig neocortex were loaded with [14C] adenosine at 37 degrees C during a 30 min incubation. After the removal of the extracellular adenosine, the preparations were superfused and adenosine derivatives were analyzed in the superfusion fluid. The measurement of the amounts of [14C] nucleotides released in the presence or absence of metabolic inhibitor allowed us to conclude that adenosine and to a smaller extent inosine, are the molecules released from the synaptosomes during superfusion.