Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP-sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors.

Reactive oxygen species signaling has been suggested to regulate stem cell development. In the present study, we treated neurospheres of rat mesencephalic precursors with inhibitors of the NADPH oxidase complex and mitochondrial ATP-sensitive potassium (mitoKATP) channel blockers during the proliferation and/or the differentiation periods to study the effects on generation of dopaminergic neurons. Treatment with low doses (100 or 250 µM) of the NADPH inhibitor apocynin during the proliferation period increased the generation of dopaminergic neurons. However, higher doses (1 mM) were necessary during the differentiation period to induce the same effect. Treatment with general (glibenclamide) or mitochondrial (5-hydroxydecanoate) KATP channel blockers during the proliferation and differentiation periods increased the number of dopaminergic neurons. Furthermore, neither increased proliferation rate nor apoptosis had a major role in the observed increase in generation of dopaminergic neurons, which suggests that the redox state is able to regulate differentiation of precursors into dopaminergic neurons.

Brain angiotensin enhances dopaminergic cell death via microglial activation and NADPH-derived ROS.

Angiotensin II (AII) plays a major role in the progression of inflammation and NADPH-derived oxidative stress (OS) in several tissues. The brain possesses a local angiotensin system, and OS and inflammation are key factors in the progression of Parkinson's disease. In rat mesencephalic cultures, AII increased 6-OHDA-induced dopaminergic (DA) cell death, generation of superoxide in DA neurons and microglial cells, the expression of NADPH-oxidase mRNA, and the number of reactive microglial cells. These effects were blocked by AII type-1 (AT1) antagonists, NADPH inhibitors, or elimination of glial cells. DA degeneration increased angiotensin converting enzyme activity and AII levels. In rats, 6-OHDA-induced dopaminergic cell loss and microglial activation were reduced by treatment with AT1 antagonists. The present data suggest that AII, via AT1 receptors, increases the dopaminergic degeneration process by amplifying the inflammatory response and intraneuronal levels of OS, and that glial cells play a major role in this process.

Functional characterization of the L-type pyruvate kinase gene glucose response complex.

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Inhibition of IkappaB kinase and IkappaB phosphorylation by 15-deoxy-Delta(12,14)-prostaglandin J(2) in activated murine macrophages.

Activation of the macrophage cell line RAW 264.7 with lipopolysaccharide (LPS) and gamma interferon (IFN-gamma) induces the expression of gene products involved in host defense, among them type 2 nitric oxide synthase. Treatment of cells with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15dPGJ(2)) inhibited the LPS- and IFN-gamma-dependent synthesis of NO, a process that was not antagonized by similar concentrations of prostaglandin J(2), prostaglandin E(2), or rosiglitazone, a peroxisomal proliferator-activated receptor gamma ligand. Incubation of activated macrophages with 15dPGJ(2) inhibited the degradation of IkappaBalpha and IkappaBbeta and increased their levels in the nuclei. NF-kappaB activity, as well as the transcription of NF-kappaB-dependent genes, such as those encoding type 2 nitric oxide synthase and cyclooxygenase 2, was impaired under these conditions. Analysis of the steps leading to IkappaB phosphorylation showed an inhibition of IkappaB kinase by 15dPGJ(2) in cells treated with LPS and IFN-gamma, resulting in an impaired phosphorylation of IkappaBalpha, at least in the serine 32 residue required for targeting and degradation of this protein. Incubation of partially purified activated IkappaB kinase with 2 microM 15dPGJ(2) reduced by 83% the phosphorylation in serine 32 of IkappaBalpha, suggesting that this prostaglandin exerts direct inhibitory effects on the activity of the IkappaB kinase complex. These results show rapid actions of 15dPGJ(2), independent of peroxisomal proliferator receptor gamma activation, in macrophages challenged with low doses of LPS and IFN-gamma.

Phorbol ester translocation of protein kinase C in guinea-pig synaptosomes and the potentiation of calcium-dependent glutamate release.

The distribution of protein kinase C activity and specific phorbol ester binding sites between soluble and particulate fractions of isolated guinea-pig cerebral cortical synaptosomes is examined following preincubation with phorbol esters. Half-maximal decrease in cytosolic activity requires 10 nM 4 beta-phorbol myristoyl acetate. Specific [3H]phorbol dibutyrate binding sites are translocated from cytoplasmic to particulate fractions in parallel with protein kinase C activity. Depolarization of the plasma membrane by 30 mM KCl does not cause translocation of protein kinase C. 1 microM 4 beta-phorbol myristoyl acetate and 1 microM 4 beta-phorbol didecanoate (but not 1 microM 4 alpha-phorbol didecanoate) enhance the release of glutamate from synaptosomes partially depolarized by 10 mM KCl; however, 4 beta-phorbol myristoyl acetate is ineffective at 20 nM. 1 microM 4 beta-phorbol myristoyl acetate slightly increases the cytosolic free Ca2+ concentration of polarized synaptosomes, but not that following partial depolarization. 4 beta-Phorbol myristoyl acetate causes a concentration-dependent increase in the Ca2+-dependent glutamate release induced by sub-optimal ionomycin concentrations, but is without effect on the release induced by maximal ionomycin. It is concluded that phorbol esters stereospecifically enhance the Ca2+-sensitivity of glutamate release, but that higher concentrations may be required than for protein kinase C translocation in the same preparation. Instead the enhancement may be related to the rapid inactivation of protein kinase C which occurs with phorbol esters.

Long-term cortical atrophy after excitotoxic striatal lesion: effects of intrastriatal fetal-striatum grafts and implications for Huntington disease.

It is not currently clear whether the cortical atrophy observed in Huntington disease (HD) is entirely a direct consequence of the disease or at least partially a secondary consequence of striatal atrophy. This is of major importance for evaluating the possible therapeutic value of intrastriatal fetal-striatum grafts in HD. Cresyl violet-stained sections from rats that had received striatal excitotoxic lesions 1 wk or 4 wk previously showed small and statistically nonsignificant decreases in the thickness of cortical layers V and VI, while series from rats lesioned 12 months previously showed marked decreases in the thickness of the whole cortex (approximately 35% decrease), layer V (approximately 45%-50%) and layer VI (approximately 45%-50%), together with marked neuron loss in these layers. In deep layer V and layer VI, Fluoro-Jade staining showed labeled neurons in animals lesioned 1 wk previously, labeled neurons and astrocytes in animals lesioned 4 wk previously, and practically no labeling in animals lesioned 12 months previously. Intracortical injection of Phaseolus vulgaris leucoagglutinin revealed that corticostriatal fibers were practically absent from the lesioned area of striata lesioned 12 months previously. However, rats that received intrastriatal fetal-striatum grafts shortly after the lesion and were killed 12 months later showed a significant reduction in cortical atrophy, and a large number of labeled corticostriatal fibers surrounding and innervating the graft. In addition, a reduction in the number of Fluoro-Jade-labeled cells in the cortex was already apparent at 3 wk post-grafting. Regardless of whether HD has a primary effect on the cortex, the present results suggest that the striatal degeneration caused by HD contributes markedly to the cortical atrophy, and that intrastriatal grafts may ameliorate this secondary component of the cortical degeneration.

Differential calcium mobilization by vasopressin, angiotensin II, gastrin-releasing peptide, and adenosine triphosphate in adult and fetal hepatocytes. Relevance for the activation of calcium-dependent enzymes.

Early signals elicited after membrane receptor binding of agonists, the transmembrane signaling pathway of which involves activation of phosphoinositide-specific phospholipase C, were compared in fetal (22 days gestation) and adult rat hepatocytes. Free cytosolic calcium changes varied depending on the agonist and type of stimulated cells. Angiotensin II and ATP elicited the maximal responses in both types of cells, whereas the maximal Ca2+ increase produced by vasopressin was twice as much in adult than in fetal hepatocytes. The opposite response was observed for bombesin- or gastrin-releasing peptide-stimulated cells. Triggering of fetal and adult hepatocytes with substances that maximally promote endoplasmic reticulum calcium release or phosphoinositide-specific phospholipase C activation revealed that at least for the actions mediated through the angiotensin II and P2 purinergic receptor, the agonist stimulation was near the maximal response capacity of the signaling pathway. Agreement was observed between the relative number of membrane receptors and the biological responses.

Substrate-dependent inhibition of protein kinase C by specific inhibitors.

Protein kinase C (PKC) and its proteolysis-derived protein kinase independent of Ca2+ and phospholipids (PKM), were purified from rat brain. By using histone H1 and protamine as substrates, we assayed the effect of several inhibitors of PKC and PKM. The inhibition turned out to be dependent on both the nature of the kinase and the type of substrate assayed. These results may help to interpret the different responses elicited by PKC inhibitors in vivo.

Striatal dopaminergic afferents concentrate in GDNF-positive patches during development and in developing intrastriatal striatal grafts.

Glial cell line-derived neurotrophic factor (GDNF) has potent trophic action on fetal dopaminergic neurons. We have used a double immunocytochemical approach with antibodies that recognize GDNF and tyroxine hydroxylase (TH) or the phosphoprotein DARPP-32, to study the developmental pattern of their interactions in the rat striatum and in intrastriatal striatal transplants. Postnatally, at one day and also at 1 week, GDNF showed a patchy distribution in the striatum, together with a high level of expression in the lateral striatal border, similar to that observed for the striatal marker DARPP-32 and also for TH. In the adult striatum, there was diffuse, weak immunopositivity for GDNF, together with widespread expression of DARPP-32-positive neurons and TH-immunoreactive (TH-ir) fibers. In 1-week-old intrastriatal striatal transplants, there were some GDNF immunopositive patches within the grafts and although there was not an abundance of TH-positive fibers, the ones that were seen were located in GDNF-positive areas. This was clearly evident in 2-week-old transplants, where TH-ir fibers appeared selectively concentrated in GDNF-positive patches. This pattern was repeated in 3-week-old grafts. In co-transplants of mesencephalic and striatal fetal tissue (in a proportion of 1:4), TH-ir somata were located mainly at the borders of areas that were more strongly immunostained for GDNF, and TH-ir fibers were also abundant in these areas and were found in smaller numbers in regions that were weakly positive for GDNF. These results demonstrate that GDNF-ir is coincident with that for TH and DARPP-32, and suggest that GDNF release by fetal striatal neurons both in normal development and in developing striatal grafts may have not only a trophic but also a tropic influence on TH-ir fibers and may be one of the factors that regulate dopaminergic innervation of the striatum.

Mechanisms of the neuroprotective effect of aspirin after oxygen and glucose deprivation in rat forebrain slices.

Acetylsalicylic acid (ASA, Aspirin) is an anti-inflammatory drug with a wide spectrum of pharmacological activities and multiple sites of action. Apart from its preventive actions against stroke due to its antithrombotic properties, recent data in the literature suggest that high concentrations of ASA also exert direct neuroprotective effects. We have used an in vitro model of brain ischaemia using rat forebrain slices deprived of oxygen and glucose to test ASA neuroprotective properties. We have found that ASA inhibits neuronal damage at concentrations lower than those previously reported (0.1-0.5 mM), and that these effects correlate with the inhibition of excitatory amino acid release, of NF-kappaB translocation to the nucleus and iNOS expression caused by ASA. All of these three mechanisms may mediate the neuroprotective effects of this drug. Our results also show that the effects of ASA are independent of COX inhibition. Taken together, our present findings show that ASA is neuroprotective in an in vitro model of brain ischaemia at doses close to those recommended for its antithrombotic effects.

Comparison between normal developing striatum and developing striatal grafts using drug-induced Fos expression and neuron-specific enolase immunohistochemistry.

The cell-level functional maturation of cell suspension grafts from embryonic day 14-15 rat striatal primordia implanted unilaterally into ibotenic acid lesioned striata of adult female rats was studied from two days to 10 weeks post-grafting. The functional and morphological characteristics of the grafts were compared with those of adult grafts (one year after implantation), normal adult striata and postnatal developing striata (up to four weeks after birth). Serial sections were stained with Cresyl Violet and investigated immunohistochemically with antibodies against dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein (DARPP-32, as a striatal marker), tyrosine hydroxylase (as a marker of dopaminergic fibres), Fos protein (as a cell-level marker of functional dopaminergic host-graft interactions), and neuron-specific enolase (correlated to differentiation and functional maturation of neuronal cells). Selected sections were double-stained for DARPP-32 and either tyrosine hydroxylase, Fos or neuron-specific enolase. The rats used to study dopamine receptor-activated expression of Fos were killed 2 h after administration of either the dopamine-releasing agent D-amphetamine (5 mg/kg intraperitoneally) or the dopamine-receptor agonist apomorphine (0.25 mg/kg subcutaneously, at which dosage it is active only on supersensitive receptors of denervated neurons). In normally developing rats, amphetamine induced Fos expression in both the striatum and globus pallidus by two weeks after birth; by four weeks, the pattern of amphetamine-induced Fos immunoreactivity was similar to that observed in adults. In the globus pallidus of both two- and three-week-old rats, amphetamine induced greater expression of Fos than in adults. Apomorphine did not induce appreciable Fos activation in either the striatum or the globus pallidus at any stage of development. In striatal grafts, amphetamine induced Fos expression from three weeks after implantation onwards, and by five to 10 weeks post-grafting the pattern of Fos immunoreactivity was similar to that observed in adult grafts. However, apomorphine induced a considerable number of Fos-positive nuclei in striatal grafts at three and four weeks after grafting. Neuron-specific enolase immunoreactivity was moderate in normal adult striatum and very high in the adult globus pallidus, and mainly located in neuronal perikarya and processes. Before two weeks of age, most neuron-specific enolase immunoreactivity was observed in internal capsule fascicles and the striatal afferents. Between two and four weeks after birth, neuron-specific enolase immunoreactivity in striatal and globus pallidus neurons gradually increased, while that in afferent fibres decreased to adult levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms of the effects of exogenous levodopa on the dopamine-denervated striatum.

The efficacy of exogenous levodopa (L-DOPA) is attributed to its conversion to dopamine by the enzyme aromatic L-amino-acid decarboxylase in striatal dopaminergic terminals. However, there is controversy about the mechanisms underlying the therapeutic and adverse effects of L-DOPA after almost all striatal dopaminergic afferents have disappeared (i.e. in the later stages of Parkinson's disease). After administration of 30mg/kg or 100mg/kg of L-DOPA, rats subjected to unilateral dopaminergic denervation showed intense contraversive rotation and a high density of Fos-immunoreactive nuclei throughout the denervated striatum, with no significant induction of Fos in the intact striatum. Injection of the central aromatic L-amino-acid decarboxylase inhibitor NSD-1015 30min before and 15min after the injection of L-DOPA suppressed the rotational behavior and the striatal induction of Fos. Comparison of results obtained in rats subjected to unilateral and bilateral dopaminergic denervation indicated that the presence of contralateral dopaminergic innervation does not significantly modulate the effects of L-DOPA on the denervated striatum. Serotonergic denervation led to slight and statistically non-significant decrease in the rotational behavior and Fos expression induced by high doses of L-DOPA (100mg/kg) in the dopamine-denervated striatum, but totally suppressed the rotational behavior and Fos expression induced by low doses of L-DOPA (30mg/kg). The present data indicate that the major effects observed after administration of exogenous L-DOPA are not due to a direct action of L-DOPA on dopamine receptors, or to extrastriatal release of dopamine, but to conversion of L-DOPA to dopamine by serotonergic terminals and probably some intrastriatal cells. Given that serotonergic neurons appear to play an important role in the action of L-DOPA in the later stages of Parkinson's disease, strategies targeting the serotonergic system should be considered for the treatment of Parkinson's disease and for combating undesirable side effects of L-DOPA therapy.

Intrathalamic striatal grafts survive and affect circling behaviour in adult rats with excitotoxically lesioned striatum.

Current models of basal ganglia disorders suggest that choreoathetosis is the end result of reduced GABAergic inhibition of the motor thalamus. Graft-derived release of GABA from intrastriatal striatal grafts has also been reported. In the present work, cell suspension grafts from embryonic day 14-15 rat striatal primordia were implanted close to the ventromedial thalamic nucleus to investigate whether they can develop and survive in this ectopic location, and whether they induce changes in the circling behaviour of the host. The grafts were implanted either in normal rats or in rats whose striatum had been lesioned with ibotenic acid. These grafts were implanted either ipsilateral or contralateral to the lesioned striatum. Additionally, some rats received intrastriatal grafts, and lesioned but non-grafted rats and lesioned rats that had received injections of saline or of cell suspensions from fetal spinal cord in the thalamus were used as control. Four to eight months after transplantation, circling behaviour after amphetamine or apomorphine injection was evaluated. Serial sections were stained with Cresyl Violet and studied immunohistochemically with antibodies against DARPP-32 (dopamine- and adenosine 3',5'-monophosphate-regulated phosphoprotein, as striatal marker), Fos protein, glutamate decarboxylase (67,000 mol. wt), glutamate decarboxylase (65,000 mol. wt) and GABA. Cresyl Violet sections showed that the intrathalamic striatal grafts developed into tissue masses resembling those observed in intrastriatal striatal grafts. DARPP-32 immunohistochemistry revealed that the grafts were composed of DARPP-32 immunoreactive (striatum-like) and DARPP-32-negative patches. The intrathalamic grafts of rats which had received a low dose of apomorphine (0.25 mg/kg) 2 h before perfusion showed clusters of intensely Fos-immunoreactive nuclei throughout the transplant, indicating that these cells had developed dopamine receptors and supersensitivity to dopamine agonists. Double Fos and DARPP-32 immunohistochemistry revealed that the Fos-positive nuclei were located in the striatum-like areas. Finally, the intrathalamic grafts also contained neurons immunoreactive to GABA and glutamate decarboxylase (65,000 and 67,000 mol. wt). Rats that had received intrathalamic grafts contralateral to the lesioned striatum (i.e. contralateral to the lesion-induced turning direction) showed a significant reduction of circling both after amphetamine (78% reduction) or apomorphine (77% reduction) injection. Rats that had received grafts ipsilateral to the lesioned striatum showed a 75% decrease in amphetamine-induced circling, but no significant change in apomorphine-induced circling. No significant drug-induced circling was observed in non-lesioned and grafted rats. Sham grafting (saline) or grafting of weakly GABAergic tissue (fetal spinal cord) had no significant effects on lesion-induced circling behaviour.(ABSTRACT TRUNCATED AT 400 WORDS)

Expression of cyclooxygenase-2 in foetal rat hepatocytes stimulated with lipopolysaccharide and pro-inflammatory cytokines.

Cyclooxygenase-2 (COX-2) is involved in the biosynthesis of prostanoids in the course of inflammatory reactions. This isoenzyme is regulated at the transcription level and many cells express COX-2 upon challenge with lipopolysaccharide (LPS) or pro-inflammatory cytokines. Since hepatocytes respond to LPS and pro-inflammatory stimuli, we investigated the expression of COX-2 in foetal and adult hepatocytes upon challenge with these substances. COX-2 was expressed in foetal hepatocytes incubated with LPS, tumour necrosis factor-alpha and interleukin-1beta. This response rapidly decreased after birth and was absent in hepatocytes from animals aged 2 days or more and treated under identical conditions. The expression of COX-2 was determined at the mRNA, protein and enzyme activity levels using Northern and Western blot, and following the synthesis of prostaglandin E2, respectively. The use of NS 398, a specific pharmacological inhibitor of COX-2, confirmed the expression of this isoenzyme in activated foetal hepatocytes. Synergism in COX-2 expression was observed between LPS, tumour necrosis factor-alpha and interleukin-1beta. Interleukin-6 and permeant analogues of cyclic AMP failed to induce COX-2 or to synergize with LPS. Also, transforming growth factor-beta inhibited the LPS- and pro-inflammatory cytokines-dependent expression of COX-2. These results indicate that foetal hepatocytes are competent to express COX-2 upon challenge with pro-inflammatory stimuli, a process lost completely in hepatocytes isolated from animals aged 2 days.

Inhibition of NOS-2 expression in macrophages through the inactivation of NF-kappaB by andalusol.

1. Andalusol, ent-6alpha,8alpha,18-trihydroxy-13(16),14-labdadiene, is a naturally occurring diterpene, isolated from Sideritis foetens (Lamiaceae). This compound exhibited therapeutic activity when evaluated in in vivo models of paw and ear inflammation (Navarro et al., 1997: Z. Naturforsch., 52, 844-849). The pharmacological effects of this diterpene have been analysed on the activation of the macrophage cell line J774 with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). 2. Incubation of J774 macrophages with andalusol (0.1 - 100 microM) inhibited the synthesis of nitrite caused by LPS (1 microg ml-1) in concentration and time-dependent manners. The maximal inhibition was observed when andalusol was added 30 min before LPS stimulation and decreased progressively as the interval between andalusol and LPS challenge increased up to 14 h. 3. Incubation of J774 cells with LPS resulted in the expression of NOS-2 protein (130 kDa) as identified by Western blot analysis. The levels of this enzyme decreased significantly in the presence of andalusol (IC50=10.5 microM), suggesting that this diterpene inhibited NOS-2 expression. 4. Andalusol inhibited nuclear factor kappaB activation, a transcription factor necessary for NOS-2 expression in response to LPS and IFN-gamma. This compound also inhibited the degradation of IkappaBalpha favouring the retention of the inactive NF-kappaB complexes in the cytosol. 5. Related compounds to andalusol but lacking the polyol groups were less effective inhibiting NOS-2 expression in LPS-activated macrophages. The present findings provide a mechanism by which the anti-inflammatory properties of this diterpene could be mediated.

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L.

Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted significant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kappaB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10-1000 ng/ml specifically promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound.

Mature intrastriatal striatal grafts revert the changes in the expression of pallidal and thalamic alpha 1, alpha 2 and beta 2/3 GABAA receptor subunit induced by ibotenic acid lesions in the rat striatum.

A between-side comparison of GABAA receptor subunit expression levels in the globus pallidus and anterior-pole motor thalamic nuclei of rats with an ibotenate lesion of the striatum, and rats receiving a fetal striatal graft in the lesioned area was made by using immunocytochemistry with subunit-specific antibodies, at different times post-lesion or different times post-grafting. At 10 days post-lesion, there was already an increase in the labeling of the alpha 1- and beta 2/3-subunits in the globus pallidus, entopeduncular nucleus and ventrolateral nucleus ipsilateral to the lesion when compared with the contralateral side, while there were no significant changes at the level of the ventromedial nucleus. Labeling of the alpha 2-subunit showed a clear increase in the entopeduncular nucleus compared with the contralateral side at 10 days post-lesion. Similar changes were also observed for the different subunits studied at 30 and 120 days after lesioning. Rats with 20-day old transplants of fetal striatal neurons that were implanted in the ibotenate lesioned striatum at 10 days post-lesioning, continued to show changes in the expression of GABAA receptor subunits, albeit at a lower level than those of ibotenate lesioned rats at similar age post-lesion. However, when examining rats with 70-day old transplants, the ibotenate-lesion induced between-side changes were almost completely compensated. These findings suggest a correlation between the maturation of the grafts and their capability to function in reestablishing neuronal circuits as shown by the reduction of changes in GABAergic transmission induced by ibotenate lesions, as indicated by the reversal of changes in GABAA receptor subunit in several areas of the basal ganglia circuit.

Time course of striatal, pallidal and thalamic alpha 1, alpha 2 and beta 2/3 GABAA receptor subunit changes induced by unilateral 6-OHDA lesion of the nigrostriatal pathway.

Immunocytochemical techniques were used to investigate the distribution and abundance of GABAA receptor subunits (alpha 1, alpha 2 and beta 2/3) in the brains of unilaterally 6-OHDA-lesioned rats. Three and 7 days after lesion, the alpha 2-subunit was significantly more abundant in the lesion-ipsilateral striatum than in the lesion-contralateral striatum; by 4 weeks after lesion, however, no significant between-side differences were observed. Three and 7 days after lesion, the alpha 1-subunit was significantly less abundant in the lesion-ipsilateral globus pallidus than in the lesion-contralateral side; again, this difference disappeared within 4 weeks of lesion. Similarly, alpha 1 was initially less abundant in several relay thalamic nuclei on the lesioned side while alpha 2 was initially more abundant in intralaminar thalamic nuclei on the lesioned side. There were no significant between-side changes for the beta 2/3-subunits. Comparison of non-lesioned and 6-OHDA-lesioned rats revealed significant differences in brain areas which also showed differences on comparison of the lesioned and non-lesioned sides of 6-OHDA-lesioned rats. These results suggest that there is an early adaptation to the lesion, achieved through changes in GABAA receptor abundance. That some of these changes are no longer apparent after 4 weeks is due not only to partial reversion of the changes in the lesioned side but also to compensatory changes in the non-lesioned side.

Effects of lesions of the nigrostriatal pathway and of nigral grafts on striatal serotonergic innervation in adult rats.

Neonatal destruction of the nigrostriatal dopaminergic system leads to serotonergic hyperinnervation of the striatum. However, it is not clear whether this occurs in adult animals. We investigated whether serotonergic sprouting occurs in adult animals, and also studied the effects of prior or subsequent implantation of dopamine-rich intrastriatal grafts. One group of adult rats received maximal 6-hydroxydopamine lesions. Other rats received maximal lesions and intrastriatal grafts 2 months later, or vice versa. The lesioned non-grafted rats showed clear serotonergic hyperinnervation throughout the striatum ipsilateral to the lesion. Intrastriatal grafts did not prevent or revert this serotonergic hyperinnervation, and were themselves densely innervated by serotonergic fibers. Serotonergic neurons usually present in the grafted cell suspension also contributed to the serotonergic innervation of the graft and the surrounding striatum.

The overall rod performance test in the MPTP-treated-mouse model of Parkinsonism.

We investigated the usefulness of the Overall Rotarod Performance (ORP) test for evaluating overall locomotory ability in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected-mouse model of Parkinson's disease (PD). For this procedure, the mice are pretrained on the rotarod and then tested at a series of increasing speeds, recording the time that the animal remains on the rod at each speed; the overall rod performance (ORP) of each animal is then calculated as the area under the curve in a plot of time-on-the-rod against rotation speed. At 15-day intervals, C57BL/6 mice were injected (or sham-injected) with MPTP, with ORP testing 7-10 days after each injection. After the fourth injection (day 45), mice in the treated group showed clearly lower ORP than mice in the control group (70-90% reduction in ORP), and were thus considered effectively lesioned. Subsequently, we investigated the short-term effects of apomorphine and L-DOPA on ORP in MPTP-treated mice. Apomorphine (at 0.5 or 2.5 mg/kg) had no significant effect, while L-DOPA (at 80 but not at 40 mg/kg) caused almost complete short-term recovery of pretreatment ORP. By about 100 days after the last MPTP injection, MPTP-treated mice showed partial long-term recovery of ORP; at this stage the mice were killed for tyrosine hydroxylase (TH) immunohistochemistry studies. TH immunoreactivity in the striatum showed a strong positive correlation with ORP as tested on day 100. We conclude that the ORP test is useful for evaluating motor deficit in MPTP-treated mice, and the effects of subsequent treatments.