17ß-Estradiol/N-acetylcysteine interaction enhances the neuroprotective effect on dopaminergic neurons in the weaver model of dopamine deficiency.

The weaver mouse, is a phenocopy of Parkinson's disease (PD) in which dopaminergic neurons degenerate gradually during development, reaching at P21 a neurodegeneration of 55%. Thus, the weaver mouse constitutes an appropriate in vivo PD model for investigating the effect of neuroprotective agents. In the present study, long-term treatment (from P1 to P21) with 17ß-estradiol (17ß-estradiol) significantly protected the dopaminergic neurons in the substantia nigra (SN) of weaver mouse by 54%, as was detected by immunohistochemical experiments, using the specific antibody against tyrosine hydroxylase (TH). This dopaminergic neuroprotection is in line with our biochemical results showing that 17ß-estradiol treatment significantly decreased the high lipid peroxidation levels seen in the SN of weaver mouse, indicating high oxidative stress. Interestingly, co-administration of 17ß-estradiol with N-acetylcysteine (NAC, precursor molecule of glutathione (GSH)) further significantly increased the survival of dopaminergic neurons in the SN (by 85%), with a parallel further decrease of lipid peroxidation to normal levels. Our results show the in vivo neuroprotective effect of 17ß-estradiol, which is strongly enhanced by co administration of NAC, indicating a strong synergistic effect of the two drugs. Furthermore, the main mechanism underlying this neuroprotective action seems to be the reversal of the oxidative stress shown by the high peroxidation levels. These results could be of clinical relevance since both drugs are already used separately in the clinic, 17ß-estradiol for treatment of PD and NAC as a mucolytic agent and for the treatment of several disorders.

Synergistic interactions of dopamine D1 and glutamate NMDA receptors in rat hippocampus and prefrontal cortex: involvement of ERK1/2 signaling.

Interactions between dopamine and glutamate receptors are essential for the prefrontal cortical (PFC) and hippocampal cognitive functions. In order to understand the molecular basis of dopamine/glutamate interactions in rat PFC and hippocampus, we investigated (a) the effect of in vitro dopamine D1 receptor stimulation on glutamate N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits' phosphorylation and (b) the signal transduction pathway underlying these interactions, by examining the involvement of D1-extracellular regulated kinase 1/2 (ERK1/2) and D1/protein kinase A (PKA)/dopamine- and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) signaling pathways. Furthermore, we compared the D1/NMDA/AMPA receptor interactions seen in PFC and hippocampus with those appearing in striatum, in which the D1 receptors' density is the highest within the mammalian brain. Our results showed that stimulation of D1 receptor by the specific agonist SKF38393 (10 microM) in PFC and hippocampal slices significantly increased the phosphorylation state of NR1ser897 and NR2Bser1303 subunits of NMDA receptor and of the GLUR1 (ser831 and ser845) subunit of AMPA receptor, as well as of ERK1/2, but not of DARPP-32. Interestingly, co-stimulation of D1 and NMDA receptors with an ineffective dose of SKF38393 (2 microM) and NMDA (5 microM) respectively, elevated further the phosphorylation level of NMDA and AMPA receptor subunits, as well as of ERK1/2, but not of DARPP-32. The D1- and D1/NMDA-induced phosphorylations were totally inhibited by SL327 (specific ERK1/2 inhibitor). Conversely, in striatal slices our data confirm that the D1-mediated phosphorylation of NMDA and AMPA receptor subunits relies on D1/PKA/DARPP-32 signaling. In conclusion, in PFC and hippocampus: (a) a strong synergistic interaction of D1 and NMDA receptors exists, which results in a significant ERK1/2 pathway activation, (b) the D1 and the D1/NMDA receptor-induced phosphorylation of NMDA and AMPA receptor subunits seems to rely on ERK1/2 signaling and could to some extent underlie the enhancement of NMDA and AMPA receptor currents mediated by D1 receptor activation.

Stimulation of adenosine A2A receptors elicits zif/268 and NMDA epsilon2 subunit mRNA expression in cortex and striatum of the "weaver" mutant mouse, a genetic model of nigrostriatal dopamine deficiency.

Interaction between basal ganglia and cerebral cortex is critical for normal goal-directed behavior. In the present study we have used the immediate early gene zif/268, as functional marker to investigate how the stimulation of adenosine A2A receptors, i.e. of the "indirect" striatal output pathway, affects striatal and cortical function in "weaver" mouse, a genetic model of dopamine deficiency. Furthermore, we have examined the effect of A2A receptor stimulation on glutamate receptor expression in the "weaver" brain. A single injection of CGS21680 (A2A receptor agonist), induced strong expression of zif/268 mRNA, detected by in situ hybridization, not only in striatum but also in the motor cortex of the "weaver" mutant. This cortical response seems to be elicited through the basal-ganglia-thalamo-cortical circuit, rather than through a direct cortical effect, since A2A receptors are not detectable in cortex according to our autoradiographic study. Co-administration of CGS21680 and quinpirole (D2 receptor agonist) attenuated the expression of zif/268 mRNA in dorsal striatum but not in motor cortex, indicating that the cortical response is dopamine-D2-receptor-independent. However, this co-administration induced an increase in zif/268 mRNA expression in somatosensory cortex, which could rely on disinhibition of the thalamo-cortical pathway. The motor cortical response could be of clinical interest, as it would further stimulate the "indirect" striatal pathway in a feed forward circuit, thus worsening the parkinsonian symptoms. Furthermore, the up-regulation of epsilon2 subunit mRNA of the NMDA receptor, induced by CGS21680 administration, seen in striatum and cortex of the "weaver" mouse, would lead to overactivity of these receptors worsening dyskinesias. These results suggest adenosine to play a significant role in regulating striatal and cortical neurochemistry in a dopamine-depleted mouse. Blockade of these receptors by specific A2A antagonists could ameliorate parkinsonian symptoms.

Brain benzodiazepine binding in aged rats.

Membrane [3H]flunitrazepam binding to central and peripheral benzodiazepine binding sites was studied in four brain areas (cerebellum, cortex, striatum and midbrain) of young (age 2-4 months) and aged (> 24 months) rats. A generalized reduction in the density of central binding sites (Bmax) was observed in all brain areas examined in aged rats. This reduction is irrelevant of the brain area and, according to literature, may correspond to cell loss and/or differential expression of mRNAs coding for the subunits of the GABA/benzodiazepine receptor complex during ageing. In the case of the peripheral binding sites, there was a decrease of Bmax in all brain areas with the exception of the cerebellum. However, the percent reduction of peripheral binding sites varied significantly among the different brain areas. These data suggest a differential effect of ageing on brain benzodiazepine binding which may reflect the special role for each brain area during ageing.

Interaction between [3H]flunitrazepam and [3H]GABA binding in the cerebellum of reeler mice.

It has been shown that in the cerebellum of reeler mutant mice GABA levels and GABA uptake increase while GABA binding decreases. This study shows that in the cerebellum of these mutants there is also an increase of benzodiazepine receptors. This increase is observed in cerebellar homogenates, in nuclei and in membranes. The increase in the density of central (i.e. clonazepam displacable) benzodiazepine receptors is primarily reflected in binding sites located in the GABA-receptor complex. In comparison to wild-type, GABA-modulin extracted from reeler cerebellum inhibits with a greater potency [3H]GABA binding. The increase in the central-type of benzodiazepine binding and its interaction with GABA binding, observed in cerebellar membranes, is interpreted as a functional response to the decrease in GABA binding and may reflect benzodiazepine receptor condensation and/or changes of subunit composition of the GABA/benzodiazepine receptor complex. The enhanced activity of reeler GABA-modulin reflects a functional response to the increased GABA levels in reeler cerebellum. The increase of the peripheral-type (i.e. PK 11195 displacable) of benzodiazepine receptors is probably due to metabolic changes that may accompany reeler cerebellar mutation. Differences in nuclear benzodiazepine binding between reeler and wild-type mice add a physiological importance to the nuclear binding of this drug.

Pentylenetetrazol-induced seizures decrease gamma-aminobutyric acid-mediated recurrent inhibition and enhance adenosine-mediated depression.

To elucidate the consequences of convulsions, we examined biochemically and electrophysiologically the brains of mice that had sustained two complete tonic-clonic convulsions after administration of pentylenetetrazol (PTZ 50 mg/kg intraperitoneally, i.p.), 48 and 24 h before decapitation. Control mice were injected with saline. Input/output curves of the extracellular synaptic responses in the CA1 area of hippocampal slices showed that PTZ-induced seizures do not establish the persistent change in hippocampal excitability itself that can be detected in vitro. However, use of the paired-pulse stimulation paradigm showed that gamma-aminobutyric acid A (GABAA)-mediated recurrent inhibition was significantly weaker (by 19-25%) in the CA1 area of slices from PTZ-treated mice (PTZ slices) as compared with slices from control mice (control slices). The density of GABAA receptors (high-affinity component) was also lower in hippocampus (by 19%) and cortex (by 14%) of PTZ-treated mice. A GABA-related disinhibitory mechanism underlying PTZ seizures may thus persist for 1 day after the seizure, predisposing the brain to subsequent seizures. On the other hand, the depressant effect of a single dose of adenosine 10 microM on the CA1 synaptic response was stronger (by 35% on population spikes) and longer lasting in PTZ slices as compared with controls. This could be attributed to significantly higher adenosine A1 receptor density in hippocampus (Bmax of [3H]CHA was higher by 34%) as well as cortex and cerebellum of these animals. The phenomenon may reflect an adenosine A1-mediated adaptive mechanism that offers protection from subsequent seizures.

Pharmacologic characterization of [3H]dopamine and [3H]spiperone binding in mouse cerebellum.

1. [3H]Dopamine and [3H]spiperone binding to cerebellar homogenates was characterized utilizing dopaminergic agonists, antagonists and non-dopaminergic drugs. 2. The [3H]DA binding to low affinity binding sites reveals a heterogeneous population consisting of dopaminergic as well as serotonergic and noradrenergic sites. However, the high affinity binding of [3H]DA reflects dopaminergic sites, although a small contribution of serotonergic and noradrenergic binding sites cannot be excluded. 3. [3H]Spiperone also labels a heterogeneous population of binding sites which, however, are mainly dopaminergic.

Cerebellar and striatal dopamine receptors: effects of reeler and weaver murine mutations.

The presence and the binding characteristics of D1 and D2 receptors were investigated in normal-reeler and normal-weaver mutant mice utilizing [3H]spiperone (D2 antagonist), [3H]SKF 38393 (D1 agonist), and [3H]DA as ligands. Analysis of the binding data showed that in the cerebellum there are two binding components for all [3H]ligands. Comparison of the binding constants from cerebellum and striatum showed that in cerebellum the high affinity-low capacity component has similar affinity with that of striatum. The reeler and weaver mutations affected the binding of all ligands: In reeler, total cerebellar specific binding sites for [3H]spiperone and [3H]SKF 38393 decrease significantly (approximately 50% and approximately 70%, respectively), while those for [3H]DA show a small (approximately 10-15%) but not significant decrease. In weaver, total cerebellar specific binding sites for [3H]spiperone, [3H]SKF 38393, and [3H]DA also decrease significantly (approximately 60%, approximately 70%, and approximately 50%, respectively). In reeler striatum [3H]SKF 38393 binding (Bmax) is significantly decreased (approximately 24%), while [3H]spiperone and [3H]DA binding (Bmax) is not affected. In weaver striatum, [3H]SKF 38393 binding is significantly increased (approximately 40%), while [3H]DA binding (Bmax) decreases significantly (approximately 70%). On the basis of the cytoarchitectural aberrations that characterize the cerebellum of these mutants and some well-established information regarding the dopaminergic system of the cerebellum, the above results indicate that in this region a) D1 receptors are mainly localized on granule cells and b) D2 receptors are localized postsynaptically on granule cells and presynaptically on the DA fibers innervating the cerebellum.

Nuclear benzodiazepine binding: possible interaction with thyroid hormone receptors.

The biochemical and pharmacological properties of nuclear [3H]flunitrazepam in brain tissues were studied. Nuclear [3H]flunitrazepam binding is saturable for both central and peripheral binding sites. Inosine and hypoxanthine displace nuclear [3H]flunitrazepam binding with greater potency than the membrane [3H]flunitrazepam binding. Triiodothyronine (T3) increases the maximum number of binding sites (Bmax) of nuclear [3H]flunitrazepam binding in vitro while thyroxine (T4) does not have any effect. Diazepam reduces the affinity of nuclear 125I-T3 binding in vitro, while the Bmax is not affected significantly. Mild digestion of chromatin, using micrococcal nuclease, reveals that a major portion of nuclear [3H]flunitrazepam binding sites are located on chromatin. These data suggest a functional role for nuclear benzodiazepine binding and a possible modulatory effect of benzodiazepines on T3 binding with its nuclear receptors.

Dopaminergic innervation and binding in the rat cerebellum.

In the present study, we used an antiserum against dopamine (DA), and specific [3H]ligands in order to shed more light on the dopaminergic system of the rat cerebellum. The immunocytochemical approach showed that the entire rat cerebellum is innervated by DA fibers. All cerebellar layers were found to receive a considerable amount of DA afferents but the molecular layer was the most heavily innervated. The analysis of [3H]DA and [3H]spiperone binding showed that in the rat cerebellum there exists DAergic binding with kinetic parameters similar to those reported for the mouse cerebellum. The results of the present study support the existence of a DA system in the rat cerebellum.

Alteration of benzodiazepine receptors in mouse cerebellum following methylazoxymethanol treatment during development.

The specific binding of [3H]flunitrazepam was studied to biochemically specify the morphological alterations induced in mouse cerebellum by a single injection of an antimitotic agent, methylazoxymethanol (MAM) performed at the beginning of the postnatal life. The MAM injection causes a general reduction of the benzodiazepine receptors in the adult mice which is particularly severe in mice having been injected the 1st day of postnatal life (so-called MAM0 mice) as compared to animals injected the 5th day (MAM5 mice): in MAM0 mice the benzodiazepine receptor is reduced to half of the control value. The affinity of the benzodiazepine towards its receptor was not affected and the topographic and biochemical action of MAM in the central nervous system was ascertained. Correlations could be made between the biochemical modifications and the morphological alterations otherwise described.

Kinetic and pharmacologic characterization of dopamine binding in the mouse cerebellum and the effects of the reeler mutation.

The purpose of this study was to characterize the dopaminergic system in the mouse cerebellum and to determine whether the dyskinesia of the reeler mutant is accompanied by alterations in cerebellar and/or striatal dopamine binding. From the analysis of (3H) dopamine ((3H)DA) and (3H)spiperone ((3H)Sp) binding, the study of the effects of several drugs on this binding, and the comparison of these parameters between the cerebellum and striatum, we conclude that a dopaminergic system exists in the cerebellum with properties common to the striatal system but also with some differences. That is, 1) with (3H)DA as ligand, we find two binding sites in cerebellum with similar Kd to those of striatum but of lower density, 2) with (3H)Sp as ligand we observe two binding sites in cerebellum and one in striatum, and 3) the competition of (3H)DA binding by various drugs shows that among the cerebellar sites, relative to striatum, there is a higher proportion that corresponds to high affinity D3 and D4 (D2 high) binding sites. In cerebellum and striatum of reeler mice, (3H)DA binding increases 125-174% and 14%, respectively.

Absence of modification in GABA and benzodiazepine binding and in choline acetyltransferase activity in brain areas of the epileptic mutant mouse tottering.

1. In the tottering mutant mouse, which suffers from epilepsy and cerebellar ataxia, we examined whether possible changes in GABA, benzodiazepine receptors and choline acetyltransferase (ChAT) activity are implicated in the pathophysiology of these animals. 2. No alteration in GABAA and GABAB binding could be detected in cerebellar membranes of epileptic mice as compared to normal mice. 3. Benzodiazepine receptor density and affinity showed no statistical difference in cerebellar membranes of epileptic and normal mice. 4. The activity of ChAT determined in the cortices of epileptic and normal mice did not differ significantly between the two groups.

Comparative aspects of cerebellar [3H]flunitrazepam and [3H]GABA binding.

[3H]Flu and [3H]GABA binding has been studied in mice and Rabbit cerebellum (Ce). The Bmax of [3H]Flu binding in Ce membranes is similar in all mice strains examined and Rabbit. However, in Ce homogenate there are significant differences in both kd and Bmax. Subcellular distribution shows higher [3H]Flu binding in the nuclear than in the membranous fraction. However, the [3H]GABA binding is lower in the nuclear than the membranous fraction.

[(3)H]GABA binding in the cerebellum of the reeler murine mutant.

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [(3)H]GABA binding have been observed. This study shows decreased affinity of the Na(+)-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na(+)-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na(+)-dependent and Na(+)-independent binding sites was not significantly altered. The decreased affinity of the Na(+)-independent GABA binding and the increased affinity of the Na(+)-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.

Characterization of nuclear triiodothyronine (T3) and tetraiodothyronine (T4) binding in developing brain tissue.

The main objective of this study was to characterize nuclear T3 and T4 binding in the developing rat brain. More specifically, we sought to determine (a) whether T3 and T4 bind to the same nuclear receptor, (b) whether there are multiple forms of nuclear T3 or T4 receptors, and (c) whether the above parameters are similar in nuclei of cerebral hemispheres and cerebellum of developing rat brain. From in vivo and in vitro binding experiments utilizing gel filtration techniques, we have shown that T3 binds to a main macromolecular fraction of molecular weight (M.W.) approx. 60 000 daltons; however, a minor binding component of M.W. greater than 100 000 daltons was also observed. Utilizing the same techniques it was shown that T4 does not bind with the main T3 binding macromolecule but only with the minor (M.W. greater than 100 000) binding component. Inasmuch as T4 competes with T3 for its binding, we have hypothesized that (a) the stability of the T4-receptor complex requires special stereochemical receptor-chromatin relationships that hold for in vivo or de novo conditions but not in the salt-extracted (0.4 M KCl) nuclear receptor preparation, or (b) T4 interacts with more than one receptor unit and forms unstable T4-receptor complexes corresponding to the high M.W. macromolecular fraction. The T3 and T4 binding characteristics described above were common to both brain regions at both developmental ages examined; however, these tissues were found to differ in quantitative aspects of T3 and T4 binding and with respect to the rate of the in vivo T4 to T3 conversion. We suggest that the nuclear T4 does not contribute to the end biological effects but, rather, it determines the number of free T3 binding sites. The end biological responses may thus be proportional to the binding of T3--derived from plasma and the local cellular conversion of T4 to T3--with its major nuclear binding protein and inversely proportional to the T4 nuclear concentration.

Studies of the catalytic properties of an endonuclease isolated from Tetrahymena pyriformis.

An acid endonuclease hydrolyzing both DNA and RNA was purified from Tetrahymena pyriformis, strain E. The enzyme is distributed in all major subcellular compartments and is excreted into the growth medium towards the middle of the logarithmic phase. It hydrolyzes DNA to penta or hexanucleotides, on the average, bearing the monoesterified phosphate at the 3'-position. Particularly in early phases of the reaction it shows a very pronounced specificity for bases with a keto group at position 4 of the pyrimidine ring, such as guanine and thymine.