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.

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.

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.

The mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate inhibits toxicity of 6-hydroxydopamine on dopaminergic neurons.

The neurotoxin 6-hydroxydopamine is commonly used in models of Parkinson's disease, and a potential factor in the pathogenesis of the disease. However, the mechanisms responsible for 6-hydroxydopamine-induced dopaminergic degeneration have not been totally clarified. Reactive oxygen species (ROS) derived from 6-OHDA uptake and intraneuronal autooxidation, extracellular 6-OHDA autooxidation, and microglial activation have been involved. The mitochondrial implication is controversial. Mitochondrial ATP-sensitive K (mitoK(ATP)) channels may provide a convergent target that could integrate these different mechanisms. We observed that in primary mesencephalic cultures and neuron-enriched cultures, treatment with the mitoK(ATP) channel blocker 5-hydroxydecanoate, inhibits the dopaminergic degeneration induced by low doses of 6-OHDA. Furthermore, 5-hydroxydecanoate blocks the 6-OHDA-induced decrease in mitochondrial inner membrane potential and inhibits 6-OHDA-induced generation of superoxide-derived ROS in dopaminergic neurons. The results suggest that low doses of 6-OHDA may generate low levels of ROS through several mechanisms, which may be insufficient to induce neuron death. However, they could act as a trigger to activate mitoK(ATP) channels, thereby enhancing ROS production and the subsequent dopaminergic degeneration. Furthermore, the present study provides additional data for considering mitoK(ATP) channels as a potential target for neuroprotection.

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.

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.

Different effects of anti-sonic hedgehog antibodies and the hedgehog pathway inhibitor cyclopamine on generation of dopaminergic neurons from neurospheres of mesencephalic precursors.

Production of dopaminergic neurons from stem/precursor cells for transplantation in Parkinson's disease has become a major focus of research. However, the inductive signals mediating this process have not been clarified. Reported data on the effects of Sonic hedgehog on differentiation of dopaminergic and serotonergic neurons from cultures of neural precursors are controversial. In the present study, cultures of proliferating neurospheres of mesencephalic precursors treated with anti-sonic hedgehog antibodies showed significantly less serotonergic and GABAergic cells and a markedly higher number of dopaminergic neurons generated from the neurospheres than control cultures. Treatment of the neurospheres with cyclopamine, which selectively inhibits sonic hedgehog signaling by preventing Smoothened activation, did not induce significant changes in generation of serotonergic and dopaminergic neurons. This suggests that Smoothened activation is not significantly involved in the above-mentioned effects and that sonic hedgehog may exert effects on the mesencephalic precursors that do not involve the canonical Patched-Smoothened-Gli signaling.

Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons.

Cell death induced by 6-hydroxydopamine (6-OHDA) is thought to be caused by reactive oxygen species (ROS) derived from 6-OHDA autooxidation and by a possible direct effect of 6-OHDA on the mitochondrial respiratory chain. However, the process has not been totally clarified. In rat primary mesencephalic cultures, we observed a significant increase in dopaminergic (DA) cell loss 24 h after administration of 6-OHDA (40 micromol/L) and a significant increase in NADPH subunit expression, microglial activation and superoxide anion/superoxide-derived ROS in DA cells that were decreased by the NADPH inhibitor apocynin. Low doses of 6-OHDA (10 micromol/L) did not induce a significant loss of DA cells or a significant increase in NADPH subunit expression, microglial activation or superoxide-derived ROS. However, treatment with the NADPH complex activator angiotensin II caused a significant increase in all the latter. Forty-eight hours after intrastriatal 6-OHDA injection in rats, there was still no loss of DA neurons although there was an increase in NADPH subunit expression and NADPH oxidase activity. The results suggest that in addition to the autooxidation-derived ROS and the inhibition of the mitochondrial respiratory chain, early microglial activation and NADPH oxidase-derived ROS act synergistically with 6-OHDA and constitute a relevant and early component of the 6-OHDA-induced cell death.

Effects of GABA and GABA receptor inhibition on differentiation of mesencephalic precursors into dopaminergic neurons in vitro.

Neurotransmitters have been shown to control CNS neurogenesis, and GABA-mediated signaling is thought to be involved in the regulation of nearly all key developmental stages. Generation of dopaminergic (DA) neurons from stem/precursor cells for cell therapy in Parkinson's disease has become a major focus of research. However, the possible effects of GABA on generation of DA neurons from proliferating neurospheres of mesencephalic precursors have not been studied. In the present study, GABA(A), and GABA(B) receptors were found to be located in DA cells. Treatment of cultures with GABA did not cause significant changes in generation of DA cells from precursors. However, treatment with the GABA(A) receptor antagonist bicuculline (10(-5) M) led to a significant increase in the number DA cells, and treatment with the GABA(B) receptor antagonist CGP 55845 (10(-5) M) to a significant decrease. Simultaneous treatment with bicuculline and CGP 55845 did not induce significant changes. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the aforementioned differences in generation of DA neurons are not due to changes in survival or proliferation of DA cells, but rather to increased or decreased differentiation of mesencephalic precursors towards the DA phenotype. The results suggest that these effects are exerted via GABA receptors located on DA precursors, and are not an indirect consequence of effects on the serotonergic or glial cell population. Administration of GABA(A) receptor antagonists in the differentiation medium may help to obtain higher rates of DA neurons for potential use in cell therapy for Parkinson's disease.

Serotonin decreases generation of dopaminergic neurons from mesencephalic precursors via serotonin type 7 and type 4 receptors.

Inductive signals mediating the differentiation of neural precursors into serotonergic (5-HT) or dopaminergic neurons have not been clarified. We have recently shown that in cell aggregates obtained from rat mesencephalic precursors, reduction of serotonin levels induces a marked increase in generation of dopaminergic neurons. In the present study we treated rat neurospheres with antagonists of the main subtypes of 5-HT receptors, 5-HT transport inhibitors, or 5-HT receptor agonists, and studied the effects on generation of dopaminergic neurons. Cultures treated with Methiothepin (5-HT(1,2,5,6,7) receptor antagonist), the 5-HT(4) receptor antagonist GR113808;67:00-.or the 5-HT(7) receptor antagonist SB 269970 showed a significant increase in generation of dopaminergic cells. Treatment with the 5-HT(1B/1D) antagonist GR 127935, the 5-HT(2) antagonist Ritanserin, the 5-HT transporter inhibitor Fluoxetine, the dopamine and norepinephrine transport inhibitor GBR 12935, or with both inhibitors together, or 5-HT(4) or 5-HT(7) receptor agonists induced significant decreases in generation of dopaminergic cells. Cultures treated with WAY100635 (5-HT(1A) receptor antagonist), the 5-HT(3) receptor antagonist Ondasetron, or the 5-HT(6) receptor antagonist SB 258585 did not show any significant changes. Therefore, 5-HT(4) and 5-HT(7) receptors are involved in the observed serotonin-induced decrease in generation of dopaminergic neurons from proliferating neurospheres of mesencephalic precursors. 5-HT(4) and 5-HT(7) receptors were found in astrocytes and serotonergic cells using double immunolabeling and laser confocal microscopy, and the glial receptors appeared to play a major role.

Angiotensin type-1-receptor antagonists reduce 6-hydroxydopamine toxicity for dopaminergic neurons.

Angiotensin II activates (via type 1 receptors) NAD(P)H-dependent oxidases, which are a major source of superoxide, and is relevant in the pathogenesis of several cardiovascular diseases and certain degenerative changes associated with ageing. Given that there is a brain renin-angiotensin system and that oxidative stress is a key contributor to Parkinson's disease, we investigated the effects of angiotensin II and angiotensin type 1 (AT(1)) receptor antagonists in the 6-hydroxydopamine model of Parkinson's disease. Rats subjected to intraventricular injection of 6-hydroxydopamine showed bilateral reduction in the number of dopaminergic neurons and terminals. Injection of angiotensin alone did not induce any significant effect. However, angiotensin increased the toxic effect of 6-hydroxydopamine. Rats treated with the AT(1) receptor antagonist ZD 7155 and then 6-hydroxydopamine (with or without exogenous administration of angiotensin) showed a significant reduction in 6-hydroxydopamine-induced oxidative stress (lipid peroxidation and protein oxidation) and dopaminergic degeneration. Dopaminergic degeneration was also reduced by the NAD(P)H inhibitor apocynin. Angiotensin may play a pivotal role, via AT(1) receptors, in increasing the oxidative damage of dopaminergic cells, and treatment with AT(1) antagonists may reduce the progression of Parkinson's disease.

Expanded mesencephalic precursors develop into grafts of densely packed dopaminergic neurons that reinnervate the surrounding striatum and induce functional responses in the striatal neurons.

The search for alternative sources of dopaminergic cells, other than primary fetal tissue for transplantation in Parkinson's disease has become a major focus of research. Different methodological approaches have led to generation in vitro of cells expressing DA-cell markers, although these cells are frequently unable to survive for a long time in vivo after transplantation and/or induce functional effects in the host brain. In the present study, we grafted cell aggregates treated with antibodies against fibroblast growth factor 4 into dopaminergic-denervated striata in rats. Furthermore, we grafted cell suspensions from primary mesencephalic fetal tissue. Grafts from expanded precursors were able to survive (at least 3 months postgrafting) and most decreased the lesion-induced ipsiversive rotation. In addition, immunolabeling for tyrosine hydroxylase and/or Fos showed that the grafts reinnervated the surrounding striatal tissue with dopaminergic terminals, and induced the expression of Fos in the striatal neurons of the reinnervated area after administration of amphetamine to the host rat. The number of dopaminergic cells in grafts from expanded precursors inducing rotational recovery was usually lower (1,226+/-314) than that in grafts from primary fetal tissue (1,671+/-122), but they were more densely packed in grafts that were of smaller volume and did not have the characteristic central nondopaminergic area observed in grafts from primary fetal tissue. The results suggest that long-term survival and functional integration into the DA-denervated striatum can be achieved with grafts of expanded mesencephalic precursors.

Angiotensin-converting enzyme inhibition reduces oxidative stress and protects dopaminergic neurons in a 6-hydroxydopamine rat model of Parkinsonism.

It is now established that the brain possesses a local renin-angiotensin system and that angiotensin II exerts multiple actions in the nervous system, including regulation of striatal dopamine release. Furthermore, angiotensin activates NADPH-dependent oxidases, which are a major source of superoxide, and angiotensin-converting enzyme inhibitors, commonly used in the treatment of hypertension and chronic heart failure, have shown antioxidant properties in several tissues. Oxidative stress is a key contributor to the pathogenesis and progression of Parkinson's disease. In the present study, we treated rats with intraventricular injections of the dopaminergic neurotoxin 6-hydroxydopamine and subcutaneous injections of the angiotensin-converting enzyme inhibitor Captopril to study the possible neuroprotective effect of the latter on the dopaminergic system and on 6-hydroxydopamine-induced oxidative stress. Rats treated with Captopril and 6-hydroxydopamine showed significantly less reduction in the number of dopaminergic neurons (i.e., immunoreactive to tyrosine hydroxylase) in the substantia nigra and in the density of striatal dopaminergic terminals than 6-hydroxydopamine-lesioned rats not treated with Captopril. In addition, Captopril reduced the levels of major oxidative stress indicators (i.e., lipid peroxidation and protein oxidation) in the ventral midbrain and the striatum of 6-hydroxydopamine-lesioned rats. Our results suggest that angiotensin-converting enzyme inhibitors may be useful for treatment of Parkinson's disease and that further investigation should focus on the neuroprotective capacity of these compounds.

Angiotensin II increases differentiation of dopaminergic neurons from mesencephalic precursors via angiotensin type 2 receptors.

In addition to the well-known actions of the humoral renin-angiotensin system, all components of this system are present in many tissues, including the brain, and may play a major role in brain development and differentiation. We investigated the possible effects of angiotensin II on the generation of dopaminergic phenotype neurons from proliferating neurospheres of mesencephalic precursors. We observed immunoreactivity for both angiotensin type 1 and type 2 (AT(1) and AT(2)) receptors in the cell aggregates. Double immunolabeling studies revealed that both receptor types are located in neurons and astrocytes. Interestingly, neurons with a dopaminergic phenotype (i.e. tyrosine hydroxylase activity) showed double labeling for AT(1) and AT(2) receptors although the labeling for AT(2) was more intense. Treatment of the neurospheres with angiotensin II (100 nm) during the differentiation period induced a marked increase (about 400%) in the generation of dopaminergic neurons. This was not affected by treatment with the AT(1) antagonist ZD 7155 but was blocked by treatment with the AT(2) antagonist PD 123319. This suggests that AT(2) receptors mediate the stimulatory effect of angiotensin II on the generation of dopaminergic neurons. Apoptotic cell death studies and bromodeoxyuridine immunohistochemistry indicated that the increase in generation of dopaminergic neurons is not due to increased survival or proliferation of dopaminergic cells during treatment with angiotensin and suggested that angiotensin induces increased differentiation of mesencephalic precursors towards the dopaminergic phenotype. Manipulation of the renin-angiotensin system may be useful for increasing production of dopaminergic neurons for transplantation in Parkinson's disease.

Interaction between the noradrenergic and serotonergic systems in locomotor hyperactivity and striatal expression of Fos induced by amphetamine in rats.

It is classically considered that Amphetamine acts by increasing extracellular dopamine levels. However, some data suggest a relevant role of other neurochemical systems. The striatum is of particular interest to the study of this question. We have investigated the involvement of the noradrenergic and serotonergic systems and their possible interaction in the striatal responses to Amphetamine using a double behavioral and immunohistochemical approach (i.e., changes in locomotor activity and striatal expression of Fos). In normal rats, Amphetamine induced locomotor hyperactivity and striatal expression of Fos. Pretreatment with the alpha1-adrenergic-receptor antagonist Prazosin or lesion of the serotonergic system significantly reduced the locomotor hyperactivity and striatal Fos expression induced by Amphetamine. Administration of Prazosin to rats with serotonergic denervation did not produce any further reduction in the Amphetamine-induced locomotor hyperactivity or striatal Fos expression compared with that observed in rats with serotonergic denervation only. Amphetamine did not induce a detectable increase in Fos expression in dopamine-denervated striata, and elicited intense rotation towards the dopamine-denervated side. This suggests that striatal dopamine release is essential in the Amphetamine-induced effects on striatal neurons. However, the noradrenergic system plays an important role, and the serotonergic system is necessary for mediating the effects of the Amphetamine-induced noradrenergic stimulation. Concurrent stimulation of dopaminergic and serotonergic receptors appears necessary to regulate Amphetamine-induced responses in the striatal neurons.

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.

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.

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.

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.

Rat brain cholinergic, dopaminergic, noradrenergic and serotonergic neurons express GABAA receptors derived from the alpha3 subunit.

In order to study the most abundant GABAA receptor subtypes expressed in cholinergic, dopaminergic, noradrenergic and serotonergic neurons (i.e., in neurons of the so-called "global" projection systems), we employed double-immunocytochemical techniques combining the labeling of GABAA receptor alpha1, alpha2 and alpha3 subunit with markers for these cells. Cholinergic neurons in the striatum, habenula, and pedunculo-pontine nucleus were immunonegative for the alpha1 subunit, and most were also alpha2-immunonegative. However, cholinergic neurons in the striatum, septum and pedunculo-pontine nucleus were alpha3 immunopositive. Dopaminergic neurons in the substantia nigra pars compacta were highly immunopositive for the alpha3, and noradrenergic neurons in the locus coeruleus were immunoreactive for the alpha3 and the alpha2-subunit; although neurons of these areas were negative for alpha1. Similarly, serotonergic neurons in raphe also showed a high level of labeling of alpha3, while there was a lack of immunoreactivity for the alpha1-subunit, and only some individual neurons were positive for the alpha2 subunit. As the presence of different alpha-subunits confers specific physiological and pharmacological properties to GABAA receptors, the abundance of receptors containing the alpha3 subunit (and the scarcity of receptor subtypes including the other alpha-subunits studied) may have important implications for the GABAergic regulation of brain "global" or "diffuse" projection systems.