José Ángel Ruiz-Ortega

Increased antiparkinson efficacy of the combined administration of VegF- and gDNF-loaded nanospheres in a partial lesion model of Parkinson's disease

Current research efforts are focused on the application of growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), as neuroregenerative approaches that will prevent the neurodegenerative process in Parkinson’s disease. Continuing a previous work published by our research group, and with the aim to overcome different limitations related to growth factor administration, VEGF and GDNF were encapsulated in poly(lactic-co-glycolic acid) nanospheres (NS). This strategy facilitates the combined administration of the VEGF and GDNF into the brain of 6-hydroxydopamine (6-OHDA) partially lesioned rats, resulting in a continuous and simultaneous drug release. The NS particle size was about 200 nm and the simultaneous addition of VEGF NS and GDNF NS resulted in significant protection of the PC-12 cell line against 6-OHDA in vitro. Once the poly(lactic-co-glycolic acid) NS were implanted into the striatum of 6-OHDA partially lesioned rats, the amphetamine rotation behavior test was carried out over 10 weeks, in order to check for in vivo efficacy. The results showed that VEGF NS and GDNF NS significantly decreased the number of amphetamine-induced rotations at the end of the study. In addition, tyrosine hydroxylase immunohistochemical analysis in the striatum and the external substantia nigra confirmed a significant enhancement of neurons in the VEGF NS and GDNF NS treatment group. The synergistic effect of VEGF NS and GDNF NS allows for a reduction of the dose by half, and may be a valuable neurogenerative/neuroreparative approach for treating Parkinson’s disease.

Interaction between the 5-HT system and the basal ganglia: functional implication and therapeutic perspective in Parkinson's disease.

The neurotransmitter serotonin (5-HT) has a multifaceted function in the modulation of information processing through the activation of multiple receptor families, including G-protein-coupled receptor subtypes (5-HT1, 5-HT2, 5-HT4–7) and ligand-gated ion channels (5-HT3). The largest population of serotonergic neurons is located in the midbrain, specifically in the raphe nuclei. Although the medial and dorsal raphe nucleus (DRN) share common projecting areas, in the basal ganglia (BG) nuclei serotonergic innervations come mainly from the DRN. The BG are a highly organized network of subcortical nuclei composed of the striatum (caudate and putamen), subthalamic nucleus (STN), internal and external globus pallidus (or entopeduncular nucleus in rodents, GPi/EP and GPe) and substantia nigra (pars compacta, SNc, and pars reticulata, SNr). The BG are part of the cortico-BG-thalamic circuits, which play a role in many functions like motor control, emotion, and cognition and are critically involved in diseases such as Parkinsons disease (PD). This review provides an overview of serotonergic modulation of the BG at the functional level and a discussion of how this interaction may be relevant to treating PD and the motor complications induced by chronic treatment with L-DOPA.

In vivo administration of VEGF- and GDNF-releasing biodegradable polymeric microspheres in a severe lesion model of Parkinson’s disease

In this work, the neuroregenerative potentials of microencapsulated VEGF, GDNF and their combination on a severely lesioned rat model were compared with the aim of developing a new strategy to treat advanced stages of Parkinsons disease. Both neurotrophic factors were separately encapsulated into polymeric microspheres (MSs) to obtain a continuous drug release over time. The regenerative effects of these growth factors were evaluated using a rotation behaviour test and quantified by the number of surviving TH+cells. The biological activities of encapsulated vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF) were investigated in HUVEC and PC12 cells, respectively. The treatment of 6-OHDA-lesioned rats with GDNF microspheres and with both VEGF and GDNF microspheres resulted in improved results in the rotation behaviour test. Both groups also showed higher levels of neuroregeneration/neuroreparation in the substantia nigra than the control group did. These results were confirmed by the pronounced TH+neuron recovery in the group receiving VEGF+GDNF-MS, demonstrating regenerative effects.

Effect of agmatine on locus coeruleus neuron activity: possible involvement of nitric oxide

To investigate whether agmatine (the proposed endogenous ligand for imidazoline receptors) controls locus coeruleus neuron activity and to elucidate its mechanism of action, we used single‐unit extracellular recording techniques in anaesthetized rats. Agmatine (10, 20 and 40 μg, i.c.v.) increased in a dose‐related manner the firing rate of locus coeruleus neurons (maximal increase: 95±13% at 40 μg). I1‐imidazoline receptor ligands stimulate locus coeruleus neuron activity through an indirect mechanism originated in the paragigantocellularis nucleus via excitatory amino acids. However, neither electrolytic lesions of the paragigantocellularis nucleus nor pretreatment with the excitatory amino acid antagonist kynurenic acid (1 μmol, i.c.v.) modified agmatine effect (10 μg, i.c.v.). After agmatine administration (20 μg, i.c.v.), dose‐response curves for the effect of clonidine (0.625 – 10 μg kg−1 i.v.) or morphine (0.3 – 4.8 mg kg−1 i.v.) on locus coeruleus neurons were not different from those obtained in the control groups. Pretreatment with the nitric oxide synthase inhibitors Nω‐nitro‐L‐arginine (10 μg, i.c.v.) or Nω‐nitro‐L‐arginine methyl ester (100 μg, i.c.v.) but not with the less active stereoisomer Nω‐nitro‐D‐arginine methyl ester (100 μg, i.c.v.) completely blocked agmatine effect (10 and 40 μg, i.c.v.). Similarly, when agmatine (20 pmoles) was applied into the locus coeruleus there was an increase that was blocked by Nω‐nitro‐L‐arginine methyl ester (100 μg, i.c.v.) in the firing rate of the locus coeruleus neurons (maximal increase 53±11% and 14±10% before and after nitric oxide synthase inhibition, respectively). This study demonstrates that agmatine stimulates the firing rate of locus coeruleus neurons via a nitric oxide synthase‐dependent mechanism located in this nucleus.

Stimulation of locus coeruleus neurons by non‐I1/I2‐type imidazoline receptors: an in vivo and in vitro electrophysiological study

Imidazoline binding sites have been reported to be present in the locus coeruleus (LC). To investigate the role of these sites in the control of LC neuron activity, we studied the effect of imidazolines using in vivo and in vitro single‐unit extracellular recording techniques. In anaesthetized rats, local (27 pmoles) and systemic (1 mg kg−1, i.v.) administrations of 2‐(2‐benzofuranyl)‐2‐imidazoline (2‐BFI), a selective I‐imidazoline receptor ligand, increased the firing rate of LC cells (maximal increase: 22 ± 5%, P<0.001 and 16 ± 7%, P<0.001 respectively). Chronic pretreatment with the irreversible monoamine oxidase inhibitor clorgyline (3 mg kg−1, i.p., every 12 h for 14 days) abolished this effect. In rat midpontine brain slices containing the LC, bath application (1 mM) of the imidazolines 2‐BFI, 2‐(4,5‐dihydroimidaz‐2‐yl)‐quinoline (BU224), idazoxan, efaroxan, phentolamine and (2‐2‐methoxy‐1,4‐benzodioxan‐2‐yl)‐2‐imidazoline (RX821002) reversibly stimulated LC cells. The maximal effect was ∼90% except for RX821002 and efaroxan which induced smaller maximal effects (∼58% and ∼35% respectively). Simultaneous application of idazoxan and 2BFI did not lead to additive effects. Bath application of the α2‐adrenoceptor antagonists, yohimbine (1–10 μM) and N‐ethoxycarbonyl‐1,2‐dihydroquinoline (EEDQ) (10 μM), failed to modify LC activity. The irreversible blockade of α2‐adrenoceptors with EEDQ (10 μM) did not alter the effect of idazoxan or that of efaroxan. Previous application of clorgyline (10 μM) did not modify the excitatory effect of 2‐BFI or efaroxan. Changes in the pH of the bathing solution (6.84–7.84) did not influence the effect caused by idazoxan. Bath application of 2‐BFI (1 mM) reversed the inhibition induced by diazoxide (300 μM), an ATP‐sensitive K+ channel opener, whereas application of glibenclamide (3 μM), an ATP‐sensitive K+ channel blocker, partially blocked the effect of 2‐BFI. This study shows that imidazoline compounds stimulate the firing rate of LC neurons. This effect is not mediated by α2‐adrenoceptors nor by I1 or I2‐imidazoline receptors but involves a different subtype of imidazoline receptor. Our results indicate that this receptor is located extracellularly and modulates ATP‐sensitive K+ channels.

The stimulatory effect of clonidine on locus coeruleus neurons of rats with inactivated α2-adrenoceptors: involvement of imidazoline receptors located in the nucleus paragigantocellularis

Abstract Clonidine stimulates locus coeruleus neurons by an α2-adrenoceptor-independent mechanism which probably involves imidazoline receptors. To study this effect, single-unit extracellular recordings in the locus coeruleus were performed in anaesthetised rats after complete, irreversible inactivation of α2-adrenoceptors by the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) (6 mg/kg i.p.; 6 h before experiments). After this pretreatment, clonidine applied into the locus coeruleus failed to produce any change in the cell firing rate. However, clonidine applied intravenously (320–2560 μg/kg), or locally (0.5–2.0 μl of 0.02 M) into the nucleus paragigantocellularis, a major locus coeruleus afferent, stimulated locus coeruleus neurons (increasing the firing rate by approximately 90%). Electrical lesions of the nucleus paragigantocellularis greatly attenuated the clonidine induced stimulation of locus coeruleus neurons ipsilateral to the lesion when applied intravenously. Blood pressure which was recorded simultaneously with cell recording, remained unaffected after clonidine administration in EEDQ pretreated rats. These results indicate that the clonidine-induced stimulation of locus coeruleus neurons is an indirect effect mediated by imidazoline receptors located on paragigantocellularis neurons projecting to the locus coeruleus.

The Role of the Subthalamic Nucleus in L-DOPA Induced Dyskinesia in 6-Hydroxydopamine Lesioned Rats

L-DOPA is the most effective treatment for Parkinsons disease (PD), but prolonged use leads to disabling motor complications including dyskinesia. Strong evidence supports a role of the subthalamic nucleus (STN) in the pathophysiology of PD whereas its role in dyskinesia is a matter of controversy. Here, we investigated the involvement of STN in dyskinesia, using single-unit extracellular recording, behavioural and molecular approaches in hemi-parkinsonian rats rendered dyskinetic by chronic L-DOPA administration. Our results show that chronic L-DOPA treatment does not modify the abnormal STN activity induced by the 6-hydroxydopamine lesion of the nigrostriatal pathway in this model. Likewise, we observed a loss of STN responsiveness to a single L-DOPA dose both in lesioned and sham animals that received daily L-DOPA treatment. We did not find any correlation between the abnormal involuntary movement (AIM) scores and the electrophysiological parameters of STN neurons recorded 24 h or 20–120 min after the last L-DOPA injection, except for the axial subscores. Nonetheless, unilateral chemical ablation of the STN with ibotenic acid resulted in a reduction in global AIM scores and peak-severity of dyskinesia. In addition, STN lesion decreased the anti-dyskinetogenic effect of buspirone in a reciprocal manner. Striatal protein expression was altered in dyskinetic animals with increases in ΔFosB, phosphoDARPP-32, dopamine receptor (DR) D3 and DRD2/DRD1 ratio. The STN lesion attenuated the striatal molecular changes and normalized the DRD2/DRD1 ratio. Taken together, our results show that the STN plays a role, if modest, in the physiopathology of dyskinesias.

Electrophysiological characterization of substantia nigra dopaminergic neurons in partially lesioned rats: Effects of subthalamotomy and levodopa treatment

Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta is the main histopathological characteristic of Parkinsons disease. We studied the electrophysiological characteristics of the spontaneous activity of substantia nigra pars compacta dopaminergic neurons in rats with a partial, unilateral, 6-hydroxydopamine lesion of the nigrostriatal pathway. In addition, the effects of subthalamotomy and prolonged levodopa treatment on the activity of dopaminergic neurons were investigated. As a result of the lesion ( approximately 50% neuronal loss), the number of spontaneously active neurons was significantly reduced. Basal firing rate, burst firing and responsiveness to intravenously administered apomorphine remained unchanged. In contrast, the variation coefficient, a measure of interspike interval regularity, was significantly increased. Ibotenic acid (10 microg) lesion of the ipsilateral subthalamic nucleus in lesioned rats did not modify the electrophysiological parameters. However, prolonged levodopa treatment (100 mg/kg/day + benserazide 25 mg/kg/day, 14 days) reversed the irregularity observed in cells from lesioned rats, while it induced an irregular firing pattern in cells from intact rats. Our results using an experimental model of moderate Parkinsons disease indicate that surviving substantia nigra pars compacta dopaminergic neurons fire irregularly. In this model, subthalamotomy does not modify the firing pattern while levodopa treatment efficiently restores normal firing of SNpc neurons and does not appear to be toxic to them.

Endocannabinoid Modulation of Dopaminergic Motor Circuits

There is substantial evidence supporting a role for the endocannabinoid system as a modulator of the dopaminergic activity in the basal ganglia, a forebrain system that integrates cortical information to coordinate motor activity regulating signals. In fact, the administration of plant-derived, synthetic or endogenous cannabinoids produces several effects on motor function. These effects are mediated primarily through the CB1 receptors that are densely located in the dopamine-enriched basal ganglia networks, suggesting that the motor effects of endocannabinoids are due, at least in part, to modulation of dopaminergic transmission. On the other hand, there are profound changes in CB1 receptor cannabinoid signaling in the basal ganglia circuits after dopamine depletion (as happens in Parkinson’s disease) and following l-DOPA replacement therapy. Therefore, it has been suggested that endocannabinoid system modulation may constitute an important component in new therapeutic approaches to the treatment of motor disturbances. In this article we will review studies supporting the endocannabinoid modulation of dopaminergic motor circuits.

INVOLVEMENT OF SUBTHALAMIC NUCLEUS IN THE STIMULATORY EFFECT OF Δ9-TETRAHYDROCANNABINOL ON DOPAMINERGIC NEURONS

The cannabinoid CB1 receptor which is densely located in the basal ganglia is known to participate in the regulation of movement. The present study sought to determine the mechanisms underlying the effect of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) on neurons in the substantia nigra pars compacta (SNpc) using single-unit extracellular recordings in anesthetized rats. Administration of Delta(9)-THC (0.25-2 mg/kg, i.v.) increased the firing rate of SNpc neurons (maximal effect: 33.54+/-6.90%, n=8) without modifying other firing parameters (coefficient of variation and burst firing). This effect was completely blocked by the cannabinoid receptor antagonist rimonabant (0.5 mg/kg, i.v.). In addition, the blockade of excitatory amino acids receptors by kynurenic acid (0.5 microM, i.c.v.) or a chemical lesion of the subthalamic nucleus (STN) with ibotenic acid abolished Delta(9)-THC effect. These results indicate that CB1 receptor activation modulates SNpc neuronal activity by an indirect mechanism involving excitatory amino acids, probably released from STN axon terminals in the SNpc.