Gene therapy approaches in the non-human primate model of Parkinson's disease.

The field of gene therapy has recently witnessed a number of major conceptual changes. Besides the traditional thinking that comprises the use of viral vectors for the delivery of a given therapeutic gene, a number of original approaches have been recently envisaged, focused on using vectors carrying genes to further modify basal ganglia circuits of interest. It is expected that these approaches will ultimately induce a therapeutic potential being sustained by gene-induced changes in brain circuits. Among others, at present, it is technically feasible to use viral vectors to (1) achieve a controlled release of neurotrophic factors, (2) conduct either a transient or permanent silencing of any given basal ganglia circuit of interest, (3) perform an in vivo cellular reprogramming by promoting the conversion of resident cells into dopaminergic-like neurons, and (4) improving levodopa efficacy over time by targeting aromatic L-amino acid decarboxylase. Furthermore, extensive research efforts based on viral vectors are currently ongoing in an attempt to better replicate the dopaminergic neurodegeneration phenomena inherent to the progressive intraneuronal aggregation of alpha-synuclein. Finally, a number of incoming strategies will soon emerge over the horizon, these being sustained by the underlying goal of promoting alpha-synuclein clearance, such as, for instance, gene therapy initiatives based on increasing the activity of glucocerebrosidase. To provide adequate proof-of-concept on safety and efficacy and to push forward true translational initiatives based on these different types of gene therapies before entering into clinical trials, the use of non-human primate models undoubtedly plays an instrumental role.

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.

Hypothyroidism is associated with increased myostatin expression in rats.

Besides its key role in the regulation of muscle growth during development, myostatin also appears to be involved in muscle homeostasis in adults, and its expression is upregulated during muscle atrophy. Since muscle physiology is greatly influenced by thyroid status, and the myostatin promoter region contains several putative thyroid hormone response elements, in the present study we examined the possible role of thyroid hormones in the regulation of myostatin gene expression. Adult male rats were made either hypothyroid or hyperthyroid by means of administration of 0.1% amino- triazole (AMT) in drinking water for 4 weeks, or daily injections of Levo-T4 (L-T4) (100 microg/rat) for 3 weeks, respectively. At the end of the treatment period, both myostatin mRNA and protein content were increased in AMT-treated rats in relation to control rats. In contrast, no changes in myostatin mRNA levels were detected in L-T4-treated rats. The role of thyroid hormones in the regulation of myostatin expression was also investigated in C2C12 cells in vitro. Treatment of C2C12 cells with thyroid hormones stimulated their differentiation into multinucleated myotubes, but did not induce any change in myostatin mRNA abundance. In all, our findings demonstrate that myostatin expression is increased in hypothyroid rats, thus supporting a possible role for this factor in the pathogenesis of the muscle loss that may occur in hypothyroidism.

Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum.

Identification of G protein-coupled receptors and their specific function in a given neuron becomes essential to better understand the variety of signal transduction mechanisms associated with neurotransmission. We hypothesized that angiotensin II type 1 (AT1) and dopamine D2 receptors form heteromers in the central nervous system, specifically in striatum. Using bioluminescence resonance energy transfer, a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors. Heteromerization did not affect cAMP signaling via D2 receptors but attenuated the coupling of AT1 receptors to Gq. A common feature of heteromers, namely cross-antagonism, i.e. the blockade of the signaling of one receptor by the blockade of the partner receptor, was tested in co-transfected cells. Candesartan, the selective AT1 receptor antagonist, was able to block D2-receptor mediated effects on cAMP levels, MAP kinase activation and ß-arrestin recruitment. This effect of candesartan, which constitutes a property for the dopamine-angiotensin receptor heteromer, was similarly occurring in primary cultures of neurons and rat striatal slices. The expression of heteromers in striatum was confirmed by robust labeling using in situ proximity ligation assays. The results indicate that AT1 receptors are expressed in striatum and form heteromers with dopamine D2 receptors that enable drugs selective for the AT1 receptor to alter the functional response of D2 receptors.

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.

Effect of iron and manganese on hydroxyl radical production by 6-hydroxydopamine: mediation of antioxidants.

6-Hydroxydopamine (6-OHDA) neurotoxicity has often been related to the generation of free radicals. Here we examined the effect of the presence of iron (Fe(2+) and Fe(3+)) and manganese and the mediation of ascorbate, L-cysteine (CySH), glutathione (GSH), and N-acetyl-CySH on hydroxyl radical (*OH) production during 6-OHDA autoxidation. In vitro, the presence of 800 nM iron increased (> 100%) the production of *OH by 5 microM 6-OHDA while Mn(2+) caused a significant reduction (72%). The presence of ascorbate (100 microM) induced a continuous generation of *OH while the presence of sulfhydryl reductants (100 microM) limited this production to the first minutes of the reaction. In general, the combined action of metal + antioxidant increased the *OH production, this effect being particularly significant (> 400%) with iron + ascorbate. In vivo, tyrosine hydroxylase immunohistochemistry revealed that intrastriatal injections of rats with 6-OHDA (30 nmol) + ascorbate (600 nmol), 6-OHDA + ascorbate + Fe(2+) (5 nmol), and 6-OHDA + ascorbate + Mn(2+) (5 nmol) caused large striatal lesions, which were markedly reduced (60%) by the substitution of ascorbate by CySH. Injections of Fe(2+) or Mn(2+) alone showed no significant difference to those of saline. These results clearly demonstrate the role of ascorbate as an essential element for the neurotoxicity of 6-OHDA, as well as the diminishing action of sulfhydryl reductants, and the negligible effect of iron and manganese on 6-OHDA neurotoxicity.

Afferent connections of the oculomotor nucleus in the chick.

Horseradish peroxidase was injected into the oculomotor nucleus of the chick in order to locate and characterize the neurons projecting to this nucleus. In the rostral mesencephalon, 120-180 neurons were labelled in the medial area of the ipsilateral nucleus campi Foreli; 190-220 in the interstitial nucleus of Cajal (most of them contralateral); and smaller numbers bilaterally in the medial mesencephalic reticular formation, the nucleus of the basal optic root complex, and the central grey matter. More caudally, numerous neurons were labelled in the contralateral abducens nucleus and the vestibular complex and a few in the nucleus reticularis pontis caudalis. Labelled neurons appeared ipsilaterally in the caudal region of the nucleus vestibularis superior and in the rostral tip of the nucleus descendens just lateral to the tractus lamino-olivaris. In the contralateral vestibular complex, a group of labelled cells observed in the dorsolateral area may be homologous to the mammalian cell group Y. At the level of the contralateral abducens nucleus, the most numerous group of cells (625-700) projecting to the oculomotor nucleus formed a lateromedial fringe that affected the nucleus tangentialis, the rostral tip of the nucleus descendens, and the ventrolateral region of the nucleus medialis. Only a few labelled neurons were seen in the contralateral nucleus vestibularis superior, the ipsilateral cell group A, and the ipsilateral nucleus vestibularis medialis.

Identification of abducens motoneurons, accessory abducens motoneurons, and abducens internuclear neurons in the chick by retrograde transport of horseradish peroxidase.

The location of the motoneurons innervating the lateral rectus, pyramidalis, and quadratus muscles of the chick has been determined by application of horseradish peroxidase (HRP) to these muscles and their nerve branches, and internuclear neurons in the abducens nucleus have been identified by injection of HRP into the oculomotor nucleus. Quantitative results were obtained by means of a semiautomatic image analyzer. Lateral rectus motoneurons were observed only in the ipsilateral principal abducens nucleus, where they numbered 500-550, and quadratus and pyramidalis motoneurons only in the ipsilateral accessory abducens nucleus. The 325-375 internuclear neurons that appeared in the principal abducens nucleus contralateral to the oculomotor nucleus injected with HRP were practically confined to the rostral two thirds of the nucleus, where they tended to surround the lateral rectus motoneurons in dorsal or lateral positions, though a minority of interneurons also mingled with the motoneurons in the center or at the medial face of the nucleus. Most interneurons were small and elongated, but a minority of larger interneurons morphologically similar to the lateral rectus motoneurons were also distinguishable. The 100-110 quadratus motoneurons and the 45-55 pyramidalis motoneurons mingled in the accessory abducens nucleus were larger than the lateral rectus motoneurons and sent their axons into the ipsilateral abducens nerve.

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.

Development of intrastriatal striatal grafts and their afferent innervation from the host.

The morphological maturation of cell suspension grafts of fetal striatal tissue (obtained from 14-15-day-old rat fetuses) was followed from two days to eight weeks after implantation into intact and ibotenic acid-lesioned striata of adult rats. The development of host afferent innervation of the grafts from the substantia nigra (tyrosine hydroxylase immunoreactive), mesencephalic raphe (serotonin immunoreactive), and the frontal cortex (anterogradely labelled with Phaseolus vulgaris leucoagglutinin) were revealed by immunohistochemistry. During the first weeks post-grafting, the striatal implants consisted of a mixture of mature- and immature-looking cell clusters. Grafts implanted into ibotenic acid-lesioned striatum grew rapidly (about five-fold) in volume over the first week. The areas of immature (probably proliferating) cells gradually disappeared, and by six to eight weeks the grafts had a fully mature appearance with patches of neurons which stained densely for DARPP-32 (i.e. were striatum-like) embedded within areas of essentially DARPP-32-negative (i.e. non-striatum-like) tissue. Peripheral clusters of grafted cells gradually intermingled with nearby areas of the surrounding lesioned host, and already by two to four days after implantation, coarse and densely immunoreactive host fibres from the substantia nigra, mesencephalic raphe and frontal cortex were present within the grafts. By four to five days the first DARPP-32-immunoreactive neurons appeared in patches within the mature portions of the grafts, and one to two days later the tyrosine hydroxylase-positive fibres began to sprout thin axons selectively within the DARPP-32-positive patches. Similarly, the serotonergic and cortical fibres in the grafts increased in number over the next two weeks, but they showed no preference for the DARPP-32-positive regions. Rich terminal networks were established by two to three weeks post-grafting, and by six to eight weeks the nigral, raphe and cortical afferents had reached terminal densities similar to those seen previously in long-term surviving grafts. Grafts implanted into dopamine-denervated hosts showed a normal morphological maturation of both DARPP-32-positive and -negative areas, although no tyrosine hydroxylase-positive innervation appeared within the grafts. Grafts implanted into non-lesioned striata did not grow beyond their initial size. The implanted cells showed less intermingling with the surrounding host striatum, thus resulting in sharply delineated graft-host borders. DARPP-32-positive patches developed, but they were smaller in size and generally present only in the most peripheral graft portions.(ABSTRACT TRUNCATED AT 400 WORDS)

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)

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.

Locomotor-activity-induced changes in striatal levels of preprotachykinin and preproenkephalin mRNA. Regulation by the dopaminergic and glutamatergic systems.

The mechanisms by which dopaminergic and glutamatergic inputs interact to regulate striatal neuropeptide expression during physiological motor activity are poorly understood. In this work, striatal expression of preprotachykinin (PPT) and preproenkephalin (PPE) mRNA was studied by in situ hybridization in rats killed 2 h after treadmill running (36 m/min for 20 min). Treadmill running induced a significant increase in the levels of both PPT (60% increase) and PPE (90% increase) mRNA in the striatum of normal rats. The increase in the level of PPT mRNA was blocked in rats previously subjected to nigrostriatal deafferentation (i.e., 6-hydroxydopamine lesion) or pretreated with D1-receptor antagonist SCH-23390 (0.1 mg/kg), the D2-receptor antagonist eticlopride (0.5 mg/kg), or the N-methyl-D-aspartate (NMDA) glutamate receptor antagonist MK-801 (0.1 mg/kg). The running-induced increase in the level of PPE mRNA was blocked in rats pretreated with SCH-23390 or MK-801. Rats subjected to nigrostriatal deafferentation or pretreated with eticlopride showed an increase in PPE mRNA levels (around 150% and 40% increase, respectively), that was enhanced by running (around 230% and 160% increase, respectively). These results suggest that locomotor activity increases, in a NMDA receptor dependent fashion, the excitatory influence of the corticostriatal glutamatergic system on the two populations of striatal projection neurons, as reflected by increases in the levels of PPT and PPE mRNA. The results obtained after dopamine depletion or injection of dopamine receptor antagonists suggest that a concomitant increase in dopamine release may enhance PPT mRNA level in striatonigral neurons via D1 receptors, and reduce PPE mRNA level in striatopallidal neurons via D2 receptors. Additionally, levels of dopamine and glutamate may be regulated by other complex indirect mechanisms.

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.

N-acetylcysteine enhances production of dopaminergic neurons from mesencephalic-derived precursor cells.

Epidermal growth-factor-responsive rat mesencephalic precursor cells incubated in differentiation media produce only a small number of dopaminergic (DA) cells. Supplementation of the differentiation medium with N-acetylcysteine (NAC) induced a marked increase (approximately 400%) in the number of tyrosine hydroxylase (TH)-positive cells. Treatment with interleukin-1 alpha also induced a significant increase (approximately 300%) in the number of TH-positive cells. However, we did not find additive effects between these drugs. The results suggest that NAC is effective in the production of DA cells from precursors, and that this may be related to enhancement of generation and/or survival of DA cells.

The retinopetal system in the rat.

After horseradish peroxidase had been applied to the ends of the optic nerves of Sprague-Dawley rats that had been sectioned at their entry into the eyeball, retrogradely labelled neurons were found in the pretectal region (mostly in the contralateral medial pretectal area) and in the ventrolateral area of the contralateral central grey matter at the level of the mesencephalic-metencephalic junction. In some cases a few weakly labelled neurons were also observed in the oculomotor or trochlear nuclei. These findings confirm the existence of a retinopetal system in this mammal.