Altered activity-rest patterns in mice with a human autosomal-dominant nocturnal frontal lobe epilepsy mutation in the ß2 nicotinic receptor.

High-affinity nicotinic receptors containing ß2 subunits (ß2*) are widely expressed in the brain, modulating many neuronal processes and contributing to neuropathologies such as Alzheimer's disease, Parkinson's disease and epilepsy. Mutations in both the α4 and ß2 subunits are associated with a rare partial epilepsy, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In this study, we introduced one such human missense mutation into the mouse genome to generate a knock-in strain carrying a valine-to-leucine mutation ß2V287L. ß2(V287L) mice were viable and born at an expected Mendelian ratio. Surprisingly, mice did not show an overt seizure phenotype; however, homozygous mice did show significant alterations in their activity-rest patterns. This was manifest as an increase in activity during the light cycle suggestive of disturbances in the normal sleep patterns of mice; a parallel phenotype to that found in human ADNFLE patients. Consistent with the role of nicotinic receptors in reward pathways, we found that ß2(V287L) mice did not develop a normal proclivity to voluntary wheel running, a model for natural reward. Anxiety-related behaviors were also affected by the V287L mutation. Mutant mice spent more time in the open arms on the elevated plus maze suggesting that they had reduced levels of anxiety. Together, these findings emphasize several important roles of ß2* nicotinic receptors in complex biological processes including the activity-rest cycle, natural reward and anxiety.

Survival and differentiation of adult rat-derived neural progenitor cells transplanted to the striatum of hemiparkinsonian rats.

We investigated the survival, distribution and differentiation capabilities of adult rat hippocampus-derived progenitor cells (AHPs) by grafting them into either the intact or dopamine (DA)-denervated adult rat striatum (ST). Furthermore, we tested the effects of the in vivo administration of retinoic acid (RA) on the differentiation of the grafted cells. AHPs, prelabeled in vitro with bromodeoxyuridine (BrdU) and primed with RA, were transplanted bilaterally into the ST of hemiparkinsonian rats. Twenty animals were divided in four groups: three groups received i.p. injections of RA (1.5 mg/kg/day) for 1, 2 or 4 weeks and one group received vehicle injections for 4 weeks. Approximately 60% of the implanted BrdU-immunoreactive (BrdU+) cells were present in either intact or lesioned ST after 5 weeks of transplantation, with a striking widespread radial distribution from the implantation site. The cells became morphologically integrated with the surrounding host tissue, with no evidence of tumor formation. Approximately 18% of the BrdU+ cells were immunoreactive for the glial precursor marker NG2 and occasionally BrdU+ cells co-expressed the neuronal marker TuJ1. This differentiation pattern was similar in the intact and DA-denervated ST. Although further research is needed to find more adequate methods to drive the differentiation of these cells toward the desired phenotypes, the survival, differentiation potential and widespread distribution throughout the ST observed in this study suggest that AHPs may be useful in treatment of degenerative disorders affecting the nervous system.

The indirect basal ganglia pathway in dopamine D(2) receptor-deficient mice.

Recent pathophysiological models of basal ganglia function in Parkinson's disease predict that specific neurochemical changes in the indirect pathway would follow the lack of stimulation of D(2) dopamine receptors. Post mortem studies of the basal ganglia in genetically modified mice lacking functional copies of the D(2) dopamine receptor gene allowed us to test these predictions. When compared with their congenic N(5) wild-type siblings, mice lacking D(2) receptors show an increased expression of enkephalin messenger RNA in the striatum, and an increased activity and expression of cytochrome oxidase I in the subthalamic nucleus, as expected. In addition, D(2) receptor-deficient mice display a reduced expression of glutamate decarboxylase-67 messenger RNA in the globus pallidus, as the basal ganglia model predicts. This reduction contrasts with the lack of change or increase in glutamate decarboxylase-67 messenger RNA expression found in animals depleted of dopamine after lesions of the mesostriatal dopaminergic system. Furthermore, D(2) receptor-deficient mice show a significant decrease in substance P messenger RNA expression in the striatonigral neurons which form the direct pathway. Finally, glutamate decarboxylase-67 messenger RNA expression in the basal ganglia output nuclei was not affected by mutations in the D(2) receptor gene, a fact that could probably be related to the absence of a parkinsonian locomotor phenotype in D(2) receptor-deficient mice. In summary, these findings provide compelling evidence demonstrating that the lack of endogenous stimulation of D(2) receptors is sufficient to produce subthalamic nucleus hyperactivity, as assessed by cytochrome oxidase I histochemistry and messenger RNA expression, and strongly suggest the existence of interactions between the basal ganglia direct and indirect pathways.

D3 receptor knockdown through antisense oligonucleotide administration supports its inhibitory role in locomotion.

To study the specific contribution of the D3 dopamine receptor in the generation of locomotor activity, total or partially dopamine-depleted rats were pretreated with an antisense oligodeoxynucleotide for the D3 receptor (D3R-as) and locomotor activity induced by apomorphine was measured. A 35.7% increase in locomotor activity was seen in the totally dopamine-depleted rats pretreated with the D3R-as, whereas the same antisense, caused a significantly greater increase in the locomotor response (95%) in the partially dopamine-depleted rats compared with control groups (pretreated with a control oligodeoxynucleotide or vehicle). In situ autoradiography for D3 receptors showed a 27% fall in the density of D3 receptors in the islands of Calleja compared with control animals. Our results seem to confirm that D3 receptors exert an inhibitory effect on locomotor activity, through the stimulation of both pre- and postsynaptic components.

Increased [125I]sulpiride binding in the subthalamic nucleus of rats with nigrostriatal lesions.

Subthalamic nucleus (STN) hyperactivity follows lesions of mesencephalic dopaminergic neurons in animal models of Parkinson's disease. The mechanism leading to sustained STN hyperactivity in parkinsonism is not well understood, but it seems not to depend on the integrity of striato-pallido-subthalamic connections (the so called indirect pathway). Sustained STN hyperactivity could result from the loss of the direct dopaminergic innervation of the STN. Here we report increased [125I]sulpiride binding in the STN of rats with 6-hydroxydopamine (6-OHDA) lesions of mesencephalic dopaminergic neurons. Furthermore, we found that chronic oral treatment with levodopa reverted the lesion-induced increase in [125I]sulpiride binding. Our results demonstrate that most STN D2-class dopamine receptors are postsynaptic to afferent dopaminergic fibers. Furthermore, they suggest that alterations of local STN dopaminergic mechanisms could play a role in the pathophysiology of parkinsonism and mediate the therapeutic/adverse effects of chronic levodopa administration.

Opposite roles of D1 and D5 dopamine receptors in locomotion revealed by selective antisense oligonucleotides.

Contralateral rotations induced by the D1-like agonist SKF 38393 in unilaterally 6-hydroxydopamine-lesioned rats were completely prevented by the administration of the D1-like antagonist SCH 23390. A similar result was obtained after intracerebroventricular administration of an antisense oligodeoxynucleotide for the D1 receptor (D1R-as). Contrariwise, administration of a D5R-as potentiated the effects of SKF 38393, showing a 60% increase in the rotational scores. Both effects were reversible upon cessation of D1R-as or D5R-as treatment and were also specific since rotational scores in rats treated with vehicle or with a randomly designed oligodeoxynucleotide were not modified. These results suggest that whereas D1 receptors play a facilitatory role in locomotion, D5 receptors exert an inhibitory effect.

Chronic levodopa is not toxic for remaining dopamine neurons, but instead promotes their recovery, in rats with moderate nigrostriatal lesions.

Orally administered levodopa remains the most effective symptomatic treatment for Parkinson's disease (PD). The introduction of levodopa therapy is often delayed, however, because of the fear that it might be toxic for the remaining dopaminergic neurons and, thus, accelerate the deterioration of patients. However, in vivo evidence of levodopa toxicity is scarce. We have evaluated the effects of a 6-month oral levodopa treatment on several dopaminergic markers, in rats with moderate or severe 6-hydroxydopamine-induced lesions of mesencephalic dopamine neurons and sham-lesioned animals. Counts of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra and ventral tegmental area showed no significant difference between levodopa-treated and vehicle-treated rats. In addition, for rats of the sham-lesioned and severely lesioned groups, immunoradiolabeling for TH, the dopamine transporter (DAT), and the vesicular monoamine transporter (VMAT2) at the striatal level was not significantly different between rats treated with levodopa or vehicle. It was unexpected that quantification of immunoautoradiograms showed a partial recovery of all three dopaminergic markers (TH, DAT, and VMAT2) in the denervated territories of the striatum of moderately lesioned rats receiving levodopa. Furthermore, the density of TH-positive fibers observed in moderately lesioned rats was higher in those treated chronically with levodopa than in those receiving vehicle. Last, that chronic levodopa administration reversed the up-regulation of D2 dopamine receptors seen in severely lesioned rats provided evidence that levodopa reached a biologically active concentration at the basal ganglia. Our results demonstrate that a pharmacologically effective 6-month oral levodopa treatment is not toxic for remaining dopamine neurons in a rat model of PD but instead promotes the recovery of striatal innervation in rats with partial lesions.

Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine.

The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.

Differential regional effects of long-term L-DOPA treatment on preproenkephalin and preprotachykinin gene expression in the striatum of 6-hydroxydopamine-lesioned rat.

The present study examined the effects of prolonged L-DOPA treatment (6 months) alone or in combination with unilateral 6-hydroxydopamine-induced lesion of the mesostriatal dopaminergic pathway on substance P and enkephalin mRNA expression in the rat neostriatum. This was done by means of quantitative in situ hybridization histochemistry. As reported previously, the unilateral dopaminergic lesion induced a significant and homogeneous decrease in striatal substance P mRNA expression and a marked increase in enkephalin mRNA expression in the ipsilateral neostriatum which was more pronounced in the dorsolateral than ventromedial part of the structure. Long-term L-DOPA treatment alone had no significant effects on the two striatal peptide mRNA levels. The chronic L-DOPA treatment in 6-hydroxydopamine-lesioned rats was found to partially reverse the lesion-induced down-regulation of substance P mRNA expression, without significantly affect the up-regulation of enkephalin when considering the neostriatum as a whole. Topographical analysis revealed that long-term L-DOPA treatment reversed, in fact, both post-lesional enkephalin and substance P responses to 6-hydroxydopamine lesion, in the ventromedial neostriatum, without significantly modified these peptide responses in the dorsolateral neostriatum. These findings provide new evidence that prolonged L-DOPA treatment differentially affects the post-lesional peptide responses in the ventromedial and dorsolateral parts of the neostriatum, suggesting regional cellular mechanisms in the neostriatum underlying the benefit and/or side-effects of L-DOPA treatment in parkinsonian patients.

Mechanism of action of clozapine-induced modification of motor behavior in an animal model of the "super-off" phenomenon.

We tested the effects of clozapine, and "atypical" neuroleptic with high affinity for the D4 (dopaminergic), and the 5-HT1c and 5-HT2 (serotonergic) receptor subtypes on locomotor activity in an animal model of Parkinson's disease showing a bimodal response curve to increasing doses of a D2 agonist. Sulpiride (D2 antagonist) and ritanserin (5-HT1c and 5-HT2 antagonist) were used for comparison. The D1 agonist SKF 38393 at a dose of 8 mg/kg significantly reversed the akinesia induced by chronic reserpine treatment (1 mg/kg for 5 days) and alpha-methyl-p-tyrosine pretreatment (300 mg/kg). In this model, the addition of a low dose of a D2 agonist, LY 171555 (quinpirole, 1 microgram/kg), inhibited the effects of SKF 38393, whereas the same drug at higher doses (5-50 microgram/kg) restored and potentiated the stimulatory response to D1 stimulation. Clozapine inhibited the inhibitory phase and potentiated the stimulatory phase of the curve. Sulpiride inhibited both phases of the dose-response curve (inhibitory/stimulatory), whereas ritanserin had no effect. We believe these results may reflect a disinhibition phenomenon possible mediated by the blockade by clozapine of a subpopulation of inhibitory, dopamine (DA) receptors belonging to the D2 "family."

Absence of neurotoxicity of chronic L-DOPA in 6-hydroxydopamine-lesioned rats.

We investigated the effects of 6 months' oral treatment with L-dihydroxy-phenylalanine (L-DOPA)/carbidopa on the remaining dopaminergic neurones of the substantia nigra pars compacta (SNC) and the ventral tegmental area (VTA) of rats with moderate or severe 6-hydroxydopamine (6-OHDA)-induced lesions and sham-operated animals. Using a radioimmunohistochemical method we counted tyrosine hydroxylase (TH)-radioimmunoreactive cells in the SNC and the VTA in emulsion-coated sections and measured the remaining surface area of both structures on autoradiograms. The sole difference observed was a significant increase of the remaining surface area of TH radioimmunolabelling in the SNC of moderately lesioned rats treated with L-DOPA/carbidopa compared with the untreated animals, while the rest of the parameters recorded, in both structures and groups of animals, were unchanged. This suggest that in vivo, this treatment is not toxic either to healthy dopaminergic neurones of the ventral mesencephalon or to those surviving after a 6-OHDA lesion.

Threshold of dopamine content and D1 receptor stimulation necessary for the expression of rotational behavior induced by D2 receptor stimulation under normo and supersensitive conditions.

We measured the minimum amount of endogenous dopamine (EDA), necessary for the expression of rotational behavior induced by D2 receptor stimulation in striatal or medial forebrain bundle (MFB) lesioned rats. We correlated these results with the minimum dose of D1 receptor agonists needed to substitute EDA in its permissive role for D2 motor effects to take place. Rats with unilateral quinolinic acid (QA) striatal or 6-hydroxydopamine (6-OHDA) MFB lesions were given increasing doses of the tyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT) in combination with a fixed dose of the D2 receptor agonist quinpirole (trans-(-)-4aR-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H-pyrazolo(3, 4-g) quinoline hydrochloride) and tested for rotational behavior. The animals were later sacrificed and striata removed; EDA was measured by high performance liquid chromatography (HPLC). Rotational responses were abolished by increasing doses of AMPT inducing a stepwise depletion of EDA. EDA content and rotational behavior to D2 stimulation showed a high degree of correlation. There was an abrupt reduction in rotational behavior at dopamine levels of 50-60% of controls in both animal models. In addition, striatal or MFB lesioned rats which were maximally depleted of dopamine by AMPT pretreatment received a fixed dose of quinpirole and then challenged with increasing doses of a D1 receptor agonist SKF 38393 ((+/-)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride). Rotational behavior was restored by SKF 38393 in both animal models in a dose-dependent fashion. Our results confirm the need for simultaneous D1/D2 stimulation in the generation of rotational behavior in both animal models. Moreover, they demonstrate the existence of a threshold level of D1 stimulation necessary to exert its permissive role on D2 mediated responses.

Dopaminergic sprouting in the rat striatum after partial lesion of the substantia nigra.

The capacity of the dopaminergic nerve system to reinnervate the denervated adult striatum was analyzed in a model of partial 6-hydroxydopamine-induced unilateral lesion of rat substantia nigra pars compacta. Sprouting of dopaminergic fibers entering the ventrolateral part of the striatum from a narrow zone of the external capsule was detected on the lesioned side 4 and 7 months, but not 10 days, after lesioning. Ultrastructural examination of the zone of sprouting revealed hypertrophic dopaminergic fibers and growth-cone-like structures, confirming the existence of an ongoing process of spontaneous regrowth of dopaminergic fibers. The identification of the factors involved in the regrowth of dopaminergic fibers may help to orientate molecular research into new treatments for Parkinson's disease.