Interdependence of the nigrostriatal dopaminergic systems on the two sides of the brain in the cat.

The release of [3H]dopamine in vivo was estimated in the left and right caudate nuclei of the cat during the continuous superfusion of the two structures with L-[3,5-3H]tyrosine by means of two "push-pull" cannulas. A lesion made in the left substantia nigra interrupted the release of [3H]dopamine in the ipsilateral caudate nucleus and was associated with a simultaneous increase in the release of [3H]dopamine on the contralateral side. The release of [3H]dopamine also decreased in the left caudate nucleus and increased in the right structure when dopamine was applied to the left substantia nigra which reduces the activity of the left dopaminergic pathway. A total of 120 estimations of the spontaneous release of [3H]dopamine were made simultaneously in the left and right caudate nuclei during periods characterized by a stable physiological state of the animals, and 76% of the estimations showed that an increase in the release of [3H]dopamine on one side corresponded to a decrease in the release of [3H]dopamine on the other side, and vice versa. These results demonstrate a close relation between the two nigrostriatal dopaminergic systems.

Plasma membrane expression of the neuronal glutamate transporter EAAC1 is regulated by glial factors: evidence for different regulatory pathways associated with neuronal maturation.

At the glutamatergic synapse the neurotransmitter is removed from the synaptic cleft by high affinity amino acid transporters located on neurons (EAAC1) and astrocytes (GLAST and GLT1), and a coordinated action of these cells is necessary in order to regulate glutamate extracellular concentration. We show here that treatment of neuronal cultures with glial soluble factors (GCM) is associated with a redistribution of EAAC1 and GLAST to the cell membrane and we analysed the effect of membrane cholesterol depletion on this regulation. In enriched neuronal culture (90% neurons and 10% astrocytes), GCM treatment for 10 days increases EAAC1 and GLAST cell surface expression with no change in total expression. In opposite, GLT1 surface expression is not modified by GCM but total expression is increased. When cholesterol is acutely depleted from the membrane by 10 mM methyl-beta-cyclodextrin (beta5-MCD, 30 min), glutamate transport activity and cell surface expressions of EAAC1 and GLAST are decreased in the enriched neuronal culture treated by GCM. In pure neuronal culture addition of GCM also increases EAAC1 cell membrane expression but surprisingly acute treatment with beta5-MCD decreases glutamate uptake activity but not EAAC1 cell membrane expression. By immunocytochemistry a modification in the distribution of EAAC1 within neurons was undetectable whatever the treatment but we show that EAAC1 was no more co localized with Thy-1 in the enriched neuronal culture treated by GCM suggesting that GCM have stimulated polarity formation in neurons, an index of maturation. In conclusion we suggest that different regulatory mechanisms are involved after GCM treatment, glutamate transporter trafficking to and from the plasma membrane in enriched neuronal culture and modulation of EAAC1 intrinsic activity and/or association with regulatory proteins at the cell membrane in the pure neuronal culture. These different regulatory pathways of EAAC1 are associated with different neuronal maturation stages.

The metabotropic glutamate receptor subtype 5 antagonist MPEP and the Na+ channel blocker riluzole show different neuroprotective profiles in reversing behavioral deficits induced by excitotoxic prefrontal cortex lesions.

Overactivation of excitatory amino acid receptors has been involved in several neurodegenerative diseases. The present study aims at investigating the potential neuroprotective action of 2-methyl-6-(phenylethylnyl)-pyridine (MPEP), a selective non-competitive antagonist of metabotropic glutamate receptor subtype 5, and 2-amino-6-trifluoro methoxy-benzothiole (riluzole), a Na+ channel blocker exhibiting anti-glutamatergic properties, on the ibotenate-induced damage to the rat medial prefrontal cortex. The neuroprotective efficacy of these compounds was assessed on the recovery from behavioral deficits induced by prefrontal cortical excitotoxic lesions in a reaction time task. MPEP (3, 10 or 30 mg/kg) or riluzole (2, 4 or 8 mg/kg) was administered i.p. 30 min before and after medial prefrontal cortex lesions. As previously found, lesions to the medial prefrontal cortex significantly altered the motor preparatory processes involved in the reaction time task. These deficits were prevented by MPEP 3 mg/kg and riluzole 2 mg/kg while higher doses of either compound were ineffective. Furthermore, the neuron-specific nuclear protein immunostaining of the lesioned cortical area in animals treated with the efficient dose of either compound revealed that MPEP reduced the volume of the lesion whereas riluzole reversed the decrease of neuronal density within the lesioned area. Altogether, these results suggest a neuroprotective action of MPEP as well as riluzole at both behavioral and cellular levels on excitatory amino acid-induced toxicity.

Neuroprotective effects of tacrolimus (FK506) in a model of ischemic cortical cell cultures: role of glutamate uptake and FK506 binding protein 12 kDa.

BACKGROUND: The mechanisms underlying the neuroprotective effects of the immunosuppressant tacrolimus, observed in vivo, remain unclear. Here we quantify these effects in vitro, and evaluate the potential involvement of the glutamate and/or immunophilin FK506 binding protein 12 kDa in tacrolimus-induced neuroprotection. METHODS: Primary cultures of neurons and astrocytes from rat cerebral cortex were subjected to transient oxygen-glucose deprivation. Neuronal injury was evaluated by cell counting after immunostaining experiments, lactate dehydrogenase release and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction. The involvement of the immunophilin FK506 binding protein 12 kDa was explored using an anti-FK506 binding protein 12 kDa antibody, (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate and rapamycin. Extracellular glutamate and glutamate uptake were respectively measured by high performance liquid chromatography and l-[3H]glutamate incorporation. RESULTS: When added during either oxygen-glucose deprivation or reoxygenation, FK506 (50-500 pM) offered significant neuroprotection. During oxygen-glucose deprivation, it was able to reverse the oxygen-glucose deprivation-induced increase in extracellular glutamate and decrease in glutamate uptake and this effect was reversed in the presence of threo-3-methyl glutamate, a specific inhibitor of glutamate transporter-1. Blocking FK506 binding protein 12 kDa inhibited the neuroprotection induced by tacrolimus added during either oxygen-glucose deprivation or reoxygenation. Tacrolimus-induced neuroprotection was also reversed in the presence of rapamycin, an immunosuppressant FK506 binding protein 12 kDa ligand devoid of neuroprotective properties and (3-3-pyridyl)-1-propyl(2 s)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidine carboxylate, a non-immunosuppressant ligand of FK506 binding protein 12 kDa, exerteing neuroprotective effects. CONCLUSION: The beneficial effects of tacrolimus during in vitro ischemia/reperfusion seem to indicate the restoration of a glutamate transporter-1-mediated activity and could be mediated by a FK506 binding protein 12 kDa pathway.

Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures.

This study described the involvement of short-term PKA, PKC or PI3K phosphorylation-mediated processes in the regulation of activity and trafficking of the excitatory amino acid transporters EAAC1, GLAST and GLT-1 endogenously expressed in neuron-enriched cultures. Glutamate uptake was dose-dependently decreased by inhibitors of protein kinase A (PKA), [N-[2-(p-bromocinnamylamino)-ethyl]-5-(isoquinolinesulfonamide)] (H89) or phosphatidylinositol 3-kinase (PI3K) (wortmannin), but not altered after protein kinase C (PKC) inhibition (staurosporine) or activation phorbol-12-myristate-13-acetate (PMA). Biotinylation and immunoblotting results (% of controls) showed that EAAC1 membrane expression was significantly decreased by H89 (71.9+/-4.7%) and wortmannin (63.3+/-20.0%) and increased by PMA (137.7+/-15.5%). H89 and PMA induced a significant decrease of the cell surface fraction of GLAST (54.0+/-34.1% and 73.3+/-14.3%, respectively) whereas wortmannin significantly increased this fraction (119.8+/-9.3%). After treatment with H89, the GLT-1 membrane level showed a two-fold increase (179.4+/-19.7%). Conversely, PMA and wortmannin induced a significant decrease of the cell surface expression of GLT-1 (49.0+/-15.4% and 40.7+/-33.7%, respectively). Confocal microscopy revealed a wortmannin-induced clustering of EAAC1 in the intracellular compartment. These data suggest that trafficking of glutamate transporters can be differentially regulated by PKA-, PKC- and PI3K-dependent signaling pathways and could therefore control total glutamate uptake activity. These processes may represent rapid adaptive responses to changes in the cellular environment, which significantly contribute to regulation of EAA transmission and further prevent possible excitotoxic events.

Cortical Regulation of Striatal Neuropeptide Y (NPY)-Containing Neurons in the Rat.

This study examined the functional relationships established by nigral, cortical, and thalamic striatal afferent pathways with neuropeptide Y (NPY)-containing neurons in the rat rostral striatum by coupling selective deafferentation procedures and NPY immunohistochemistry. Previous experiments have shown that after unilateral 6-hydroxydopamine (6-OHDA)-induced degeneration of nigrostriatal dopaminergic neurons, the mean number of NPY-immunoreactive (Ir) neurons per frontal section was increased in the striatum ipsilateral to the lesion side and unaltered in the contralateral striatum. The present topographical analysis of the 6-OHDA lesion effects led us to state that the increase in NPY-Ir neuron density occurs in restricted ventral and medial zones of the ipsilateral striatum. Unilateral ablation of the frontoparietal cerebral cortex by thermocoagulation was moreover shown to elicit, 20 - 30 days later, a significant bilateral increase in the number of striatal NPY-Ir cells. The increase was more marked in the striatum ipsilateral to the hemidecortication where it was similar in amplitude to that induced by the 6-OHDA lesion. The topographical analysis of the cortical lesion effects also revealed an uneven striatal response, but, in contrast to that observed for the 6-OHDA lesion, changes were restricted to dorsolateral areas of the striatum in both brain sides, revealing an apparent complementarity of nigral dopaminergic and cortical influences over striatal NPY neuronal system. Combined unilateral nigral and cortical lesions surprisingly counteracted in a survival time dependent manner the effects of each lesion considered separately. In that condition topographical changes related to the 6-OHDA lesion totally disappeared and those related to the cortical lesion were attenuated but still present. These results suggest that expression of striatal dopamine - NPY interaction is dependent on corticostriatal transmission. Interestingly lesion of thalamic areas projecting to the striatum did not significantly modify the mean number of NPY-Ir neurons determined per section from the whole striatal surface, but selectively increased the NPY neuron density in the mediodorsal region of the striatum, suggesting that the striatal NPY-containing neuronal system is also influenced by thalamostriatal projections.

Striatal NPY-Containing Neurons Receive GABAergic Afferents and may also Contain GABA: An Electron Microscopic Study in the Rat.

Dual labelling methods were applied to localize simultaneously neuropeptide Y (NPY) and glutamate decarboxylase (GAD) immunoreactivities on ultrathin sections of the rat caudate-putamen (CP). By means of a double peroxidase-anti-peroxidase technique, using 3,3'-diaminobenzidine and benzidine dihydrochloride as chromogens in animals with no colchicine pretreatment, GAD immunoreactivity was found to be present in terminals only whereas NPY immunoreactivity was detected in neurons displaying the features of aspiny type cells and processes. With this approach, we observed numerous synaptic associations of the symmetrical type between GAD-immunoreactive (-Ir) axonal boutons and NPY-Ir cell bodies and dendrites. By combining immunoperoxidase and radioimmunocytochemical labelling in animals pretreated with colchicine, NPY was again detected in a single population of aspiny type neurons whereas GAD immunoreactivity was observed in neurons which could be classified as aspiny and spiny on the basis of their ultrastructural characteristics. All the cells of the aspiny type displaying clear-cut NPY immunoreactivity were also found to be GAD-positive. Some other neurons of both the aspiny and the spiny type were found to be immunoreactive to GAD alone. GAD/NPY dually labelled terminals were also observed and some axo-axonic appositions between GAD- and NPY-Ir terminals were also detected. All in all, these data show that NPY aspiny type neurons of the rat CP receive GABAergic afferents and provide morphological support for two hypotheses: that NPY is co-localized with GABA in some cell bodies, dendrites and axons, and that presynaptic interactions may occur between NPY and GABAergic neuronal systems.

Effects of pyroglutamic acid on corticostriatal glutamatergic transmission.

The effects of L-pyroglutamic acid, a molecule structurally derived from L-glutamic acid (Glu), were measured on the high affinity of uptake of glutamic acid from striatal synaptosomes of the rat and on the binding of [L-3H]glutamic acid to striatal membranes. The results showed a competitive inhibition of the high affinity transport of glutamic acid by L-pyroglutamic acid in vitro with no effect on the uptake of gamma-aminobutyric acid (GABA). An inhibition of the binding of [L-3H]glutamic acid to striatal membranes was also detected. Significant high affinity uptake of [L-3H]pyroglutamic acid was evident in synaptosomes from the striatum. A regional distribution study of the uptake processes for [L-3H]glutamic acid and [L-3H]pyroglutamic acid in different areas of the brain showed a similar distribution, suggesting that an uptake of [L-3H]pyroglutamic acid, although weak, occurs in glutamatergic nerve terminals. This proposal was further reinforced by measuring the effects of a large cortical lesion involving frontal and parietal areas on the uptake of [L-3H]glutamic acid and [L-3H]pyroglutamic acid in synaptosomes from the striatum. The results showed a large decrease in the uptake processes of both labelled molecules showing that the uptake of [L-3H]pyroglutamic acid, as for glutamic acid mainly occurred in corticostriatal nerve terminals, although other uptake sites are not excluded.

Regional distribution and ontogeny of 5-HT4 binding sites in rodent brain.

We have investigated the regional distribution of 5-hydroxytryptamine4 (5-HT4) receptor binding sites in the adult guinea pig, rat and mouse brain using the specific 5-HT4 antagonist [3H]GR113808 as a radioligand. The developmental changes in the expression of these binding sites were also investigated quantitatively in the rat brain (gestational days 16 and 19; postnatal days 1, 3, 7, 9, 12 and 21). In order to compare previously obtained data on primary cultures, semi-quantitative analysis was also performed during mouse brain ontogeny (postnatal days 1, 7 and 11). The main finding of this study is that 5-HT4 receptors have comparable, wide and heterogeneous distributions in the adult brain of the species investigated, with densities reaching adult levels between the second and third postnatal week in most regions of the rat and mouse brain. In contrast, a progressive loss of 5-HT4 binding sites is observed in the pons, whereas a transient peak of receptor expression is seen during the second postnatal week in the globus pallidus and substantia nigra pars lateralis. The developmental pattern of 5-HT4 receptor distribution suggests, except in latter regions, that these receptors probably exert a minor role in developmental processes. In the adult, high densities of [3H]GR113808 binding sites are present in various regions belonging to limbic system (islands of Calleja, olfactory tubercle, fundus striati, ventral pallidum, septal region, hippocampus, amygdala), or known to be components of different pathways, such as the hippocampo-habenulo-interpeduncular and the striato-nigro-tectal pathways. While the regional distributions of [3H]GR113808 binding sites were identical in the mouse and rat, some differences were observed in the guinea-pig, in particular in the globus pallidus, substantia nigra and interpeduncular nucleus. The expression of 5-HT4 receptors in limbic areas is highly suggestive of a role for these receptors in emotional processes, whereas their expression in the striato-nigral-tectal pathway might be indicative of a role in the control of visuo-motor activity.

Activation of the adenylate cyclase-dependent protein kinase pathway increases high affinity glutamate uptake into rat striatal synaptosomes.

This study examined the effects of various agents known to alter protein phosphorylation through protein kinase A or C on high affinity glutamate uptake measured in vitro on rat striatal homogenates. Incubation of synaptosomes with the phosphatase inhibitor, okadaic acid, dramatically increased glutamate uptake indicating that underlying phosphorylation mechanisms may be involved in the regulation of this transport process. The protein kinase C activator, phorbol-12,13-dibutyrate, or inhibitor, staurosporine, did not significantly modify glutamate uptake. In contrast, forskolin, which activates adenylate cyclase, induced a dose-dependent increase in glutamate uptake. Saturation kinetic analysis indicated that forskolin increased the Vmax without modifying the Km of the transport process as compared to control values. The effect of forskolin was mimicked by dibutyryl adenosine monophosphate, an analog of cAMP which activates protein kinase A, and counteracted by high doses of N-[2-(methylamino) ethy1]-5-isoquinoline sulfonamide, a protein kinase A inhibitor. These results suggest that an adenylate cyclase-dependent protein kinase may be involved in the post-translational regulation of glutamate transporters.

N-methyl-D-aspartate receptor blockade impairs behavioural performance of rats in a reaction time task: new evidence for glutamatergic-dopaminergic interactions in the striatum.

The effects of blocking glutamate transmission at the N-methyl-D-aspartate receptor subtype were studied in rats performing a conditioned reaction time motor task. Rats were trained to release a lever after the onset of a visual stimulus within a time limit to obtain food reward. The results showed that the performances of the groups receiving the N-methyl-D-aspartate receptor antagonists dizocilpine maleate (0.1 mg/kg) injected systemically or DL-2-amino-5-phosphonovaleric acid at the highest dose tested (5.0 micrograms/microliter/side) injected locally into the striatum changed significantly as compared to controls. The effects of these antagonists, consisting of an increase in the number of lever releases occurring before the visual stimulus onset ("anticipated responses"), were similar to those induced by injecting dopamine into the same striatal location. Both dizocilpine maleate and DL-2-amino-5-phosphonovaleric acid (5.0 micrograms/microliter) reversed the motor deficits, resulting in an increase in the number of lever releases after the time limit ("delayed responses") that were induced by the D2 dopamine receptor antagonist raclopride. Although these results partly confirm the existence of a functional antagonism between the glutamatergic and the dopaminergic systems in the striatum, opposite findings were obtained with the group that received intrastriatal DL-2-amino-5-phosphonovaleric acid at the lowest dose (0.5 micrograms/microliter/side). When given alone, 0.5 micrograms/microliter DL-2-amino-5-phosphonovaleric acid had no behavioural effects, but when jointly administered with dopamine or raclopride, it was found to reverse the effects of dopamine and to potentiate the motor deficits induced by raclopride. These opposite effects on the reaction time task observed after the intrastriatal injection of DL-2-amino-5-phosphonovaleric acid, depending on the dose tested, occurred only after a combined treatment with a dopaminergic agonist or antagonist and suggest that the level of the striatal dopaminergic activity may play a critical role in regulating the glutamate transmission via the N-methyl-D-aspartate receptors during the performance of complex sensorimotor tasks of this kind.

Dopamine and complex sensorimotor integration: further studies in a conditioned motor task in the rat.

Rats were trained to depress a lever and wait for the onset of a light stimulus, occurring after four equiprobable and variable intervals. At the stimulus onset, they had to release the lever within a reaction time limit for food reinforcement. This paradigm required time estimation of the various intervals and high attentional load for correct performance. Following activation of the dopaminergic transmission after systemic injection of d-amphetamine (0.6 and 0.8 mg/kg) or intrastriatal injection of dopamine (2.5 microgram/microliters), the rat's performance was impaired. Compared with control animals, the performance deficits were expressed as an increased number of premature lever releases before the conditional stimulus onset ("premature responses") and decreased reaction times. Indeed, the reaction times distribution was shifted to the left towards shortened reaction times. Although the number of premature responses was increased, the time estimation of the four different equiprobable intervals was not disturbed after stimulation of dopaminergic activity. A delay-dependent shortening of reaction times as a result of the conditional probability of the stimulus occurrence (i.e. reaction times are shorter as the duration of the delay increases) was found in control and drug sessions, indicating that the animals were still able to prepare their motor response (lever release) even after overstimulation of the dopaminergic transmission. In contrast, blocking dopamine receptors with the selective D2 antagonist raclopride was found to induce opposite effects on the reaction time performance. The number of delayed responses (i.e. occurring with a latency > 600 ms) was found to be significantly enhanced. Furthermore, the reaction times distribution showed a shift of the values to the right revealing a general tendency to lengthened reaction times. These results indicate that a "critical level" of dopamine activity (neither too low nor too high) in the striatum is necessary for a correct execution of the movement in a conditioned motor task with temporal constraint. Moreover, while delayed responses might reflect a motor impairment, anticipatory responses might reflect a "motor facilitation" revealed by a higher level of motor readiness, without disturbing time estimation nor attentional processes.

Early and widespread normalization of dopamine-neuropeptide Y interactions in the rat striatum after transplantation of fetal mesencephalon cells.

Graft-to-host interactions were examined at cellular level, by measuring changes in the immunoreactivity of striatal interneurons expressing neuropeptide Y after dopamine denervation and transplantation of fetal mesencephalon neurons into the striatum of adult rats. Mesencephalic cell suspensions were implanted unilaterally into the dorsal part of the striatum in rats two weeks after intranigral injection of 6-hydroxydopamine. One month and three to four months later, rats showing abolition of amphetamine-induced turning were perfused. Serial brain sections containing intrastriatal grafts were treated for tyrosine hydroxylase and neuropeptide Y immunocytochemistry, and neuropeptide Y-immunoreactive neurons were quantified in various parts of the striatal surface and compared with the striatum of controls and age-matched rats with lesions. Biochemical analyses of dopamine and dihydroxyphenyl acetic acid tissue levels and [3H]dopamine uptake were also performed on striatal samples from similar groups of normal, lesioned and transplanted rats. As early as one month post-grafting, a complete reversal of the increase in the number of neuropeptide Y-immunoreactive neurons occurring after 6-hydroxydopamine lesion was observed in dopamine-grafted animals, although a partial restoration of the tyrosine hydroxylase immunostaining and a recovery of 8% dopamine tissue level were observed in the striata of grafted as compared to normal rats. This effect on the host immunoreactivity was found to be specific to dopamine grafts, since no reversal was observed in sham-spinal cord-transplanted rats. Moreover, similar degrees of normalization were recorded either in the total striatum, or in the area immediately adjacent to the graft, or even in the zone most sensitive to dopamine denervation in terms of neuropeptide Y immunoreactivity. No more pronounced functional effects were observed three to four months after transplantation. These data suggest that grafted dopamine neurons are able to induce rapid and extensive host responsiveness, possibly by means of mechanisms involving synaptic and diffuse release of dopamine and adaptive changes in the host brain. These data may provide a cellular basis for interpreting larger behavioural recoveries than those expected to occur with dopamine grafts in view of the partial restoration of the dopaminergic innervation.

Pharmacological characterization of dopaminergic influence on expression of neuropeptide Y immunoreactivity by rat striatal neurons.

Selective unilateral lesion of the nigrostriatal dopamine pathway by the cytotoxin 6-hydroxydopamine was previously shown to enhance the number and staining intensity of neurons expressing neuropeptide Y immunoreactivity in the ipsilateral striatum. This effect was completely reversed by treatment of the 6-hydroxydopamine-injected animals with the directly acting dopamine agonist apomorphine. This finding reinforces our previous hypothesis that changes in striatal neuropeptide Y staining subsequent to 6-hydroxydopamine lesions of this kind reflect changes in intraneuronal neuropeptide Y levels which are directly attributable to the suppression of a tonic dopaminergic control. In contrast to the effect of 6-hydroxydopamine lesion, non-destructive impairment of striatal dopamine transmission by treatments with either the dual dopamine D1/D2 receptor antagonist haloperidol or the dopamine synthesis inhibitor alpha-methylparatyrosine induced a decrease in both the number of neuropeptide Y striatal cells (-29.8% and -34.8%, respectively) and in their labeling intensity. The selective D2-antagonist sulpiride also showed a tendency to reduce the number of neuropeptide Y immunoreactive cells, whereas the selective D1 antagonist SCH 23390 induced a small but constant increase in this number. Taken as a whole, these results suggest that the dopaminergic D1 and D2 receptor subtypes play opposite roles in the dopaminergic control of the striatal neuropeptide Y neuronal system, which may account for the different changes in striatal neuropeptide Y immunostaining observed after 6-hydroxydopamine injury and after non-destructive impairment of nigrostriatal dopaminergic transmission.

Reversal of the adaptive response of neuropeptide Y neurons in the rat striatum to nigrostriatal dopamine deafferentation by the N-methyl-D-aspartate antagonist dizocilpine maleate.

This study examined the effects of systemic treatments with dizocilpine maleate alone or in combination with unilateral 6-hydroxydopamine-induced lesion of the nigrostriatal dopaminergic neurons on the number and staining intensity of neuropeptide Y-immunoreactive neurons in the rat striatum. In the combined condition, short-term and long-term treatments with dizocilpine maleate were started 19 days and 12 days after the lesion of the nigrostriatal dopaminergic pathway, respectively. As reported previously, the unilateral dopaminergic lesion elicited an increase in both the number and staining intensity of neuropeptide Y-immunoreactive neurons in the ipsilateral striatum. Short-term treatment with dizocilpine maleate at the dose of 0.2 mg/kg (four injections, 6 h apart, sacrifice 2 h after the final dose), which by itself did not modify neuropeptide Y immunostaining, totally suppressed the effect of the dopaminergic deafferentation on the number of neuropeptide Y-positive neurons but not that on the intraneuronal amount of labelling. When administered twice a day for eight days at the same dose of 0.2 mg/kg, dizocilpine maleate by itself elicited an increase in the number of neuropeptide Y-immunodetectable cells, paradoxically concomitant with a decrease in the levels of intraneuronal labelling. After combination of this treatment with unilateral lesion of the nigrostriatal dopaminergic pathway, the changes related to either the dizocilpine maleate treatment or the 6-hydroxydopamine-induced lesion totally disappeared, so that the number and staining intensity of neuropeptide Y-immunoreactive neurons in that condition did not differ from control values.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural recovery of rats grafted with dopamine cells after partial striatal dopaminergic depletion in a conditioned reaction-time task.

The functional effects of grafts of dopamine-rich ventral mesencephalic suspension transplanted in a partially dopamine-depleted striatum were studied in rats performing a reaction-time motor task. The animals were trained to depress a lever, hold it down and release it within a limited period of time (700 ms) after the onset of a visual conditioned stimulus to obtain a food reward. The animals' performances were tested daily for up to two months after transplantation and for up to three months in the case of the animals with lesion only (bilateral striatal 6-hydroxydopamine injection). The baseline performances of the sham-operated control animals tended to improve, whereas the performances of the lesioned rats were significantly disrupted throughout the three months test. The majority of the animals (13/21) in the lesion group showed severe deficits mainly reflected in an increase in the number of the anticipated responses (premature release of the lever before the visual stimulus), and also in the number of the delayed responses (lever release after the time limit) recorded after dopamine depletion. The remaining animals (8/21) exhibited mild deficits (delayed responses only). These differences in the performance deficits appeared to be in relation to the extent of the dopamine denervation within the striatum assessed by the tyrosine hydroxylase immunostaining. Grafted animals showed a large number of dopamine fibers in the reinnervated striata and most of them (73%) significantly improved the reaction-time performance after transplantation. In the most severely impaired animals the number of anticipated errors was totally reversed within one month post-grafting, while the number of delayed responses remained high after transplantation. The performances of the less severely impaired animals returned more rapidly (within three weeks) to the pre-operative levels. The results show that intrastriatal ventral mesencephalic transplants are able to induce substantial or complete recovery in a complex reaction-time task. In the present model for partial dopamine depletion of the striatum, the mechanisms underlying the graft-induced recovery probably involve the participation of endogenous dopamine neurons acting in addition to, and/or in synergy with the dopamine-rich grafted tissue so that a functional level of dopaminergic transmission is restored in transplanted animals.

Motor impairments and neurochemical changes after unilateral 6-hydroxydopamine lesion of the nigrostriatal dopaminergic system in monkeys.

Unilateral lesions of the nigrostriatal dopaminergic system were induced in five monkeys by intranigral injections of the neurotoxin 6-hydroxydopamine. Following the lesion, all monkeys showed a transient reluctance in using the contralateral forelimb, accompanied, in two monkeys by semi-flexed posture of the disabled forelimb. Three of the monkeys that had been conditioned to perform a visually triggered goal-directed arm movement, showed an increase in latency and duration of contralateral arm movements. Task performance recovered spontaneously to preoperative levels within four months in two monkeys despite significant reductions of endogenous dopamine and dihydroxyphenylacetic acid contents in the caudate nucleus, putamen and globus pallidus ipsilateral to the neurotoxic nigral injection. The third monkey exhibited a persistent increase in movement latency associated with a near complete loss of dopamine in both the putamen and the caudate nucleus. In all cases, an increase the dihydroxyphenyl-acetic acid to dopamine ratio was detected in the striatum and pallidum suggesting a compensatory increase in dopamine turnover in remaining intact dopaminergic nerve terminals. The level of serotonin was changed in all monkeys consisting of either a decrease or an increase, depending on the striatopallidal regions studied. Changes in choline acetyltransferase and glutamic acid decarboxylase activities in the same regions were only seen in some cases. The present results show that 6-hydroxydopamine-induced partial unilateral lesion of nigral dopaminergic neurons produced predominantly contralateral hypokinesia, accompanied by reductions of dopamine content in the ipsilateral striatum and pallidum. The use of this locally applied neurotoxin appears to be a suitable method for investigating neurophysiological mechanisms underlying hypokinesia since deficits in both initiating and executing movements can be expressed independently of other behavioral symptoms. The results show more persistent deficits in starting movements than in their execution and thus suggest that motor initiation is more dependent upon the functional integrity of the nigrostriatal dopamine system than movement completion.

Ultrastructural relationships between choline acetyltransferase- and neuropeptide y-containing neurons in the rat striatum.

The relationships between cholinergic and neuropeptide Y-containing neuronal systems in the rat striatum were examined using a dual immunoperoxidase labelling method. These neurons were identified by their immunoreactivity to choline acetyltransferase and neuropeptide Y, respectively, and were visualized on the same sections using 3,3'-diaminobenzidine and benzidine dihydrochloride as distinct chromogens under two conditions: (i) neuropeptide Y detection by the 3,3'-diaminobenzidine diffuse brown reaction product and choline acetyltransferase detection by the benzidine dihydrochloride blue, granular reaction product; (ii) choline acetyltransferase detection by 3,3'-diaminobenzidine and neuropeptide Y detection by benzidine dihydrochloride. Although both neuropeptide Y- and choline acetyltransferase-immunoreactive cell bodies were simultaneously detected and were easily distinguishable whatever the conditions used, neuropeptide Y- and choline acetyltransferase-immunoreactive dendrites and axons could not be visualized on the same sections, since only the diaminobenzidine-labelled processes were detectable. Light microscopic observations on sections dual labelled with either method confirmed that choline acetyltransferase and neuropeptide Y immunoreactivities were localized in morphologically different populations of striatal neurons scattered throughout the striatum, choline acetyltransferase immunoreactivity being associated with large neurons and neuropeptide Y immunoreactivity with medium-sized neurons. In addition, the choline acetyltransferase-immunoreactive neurons were found to be more numerous than the neuropeptide Y-immunoreactive neurons and to be prevalent in the dorsolateral areas of the striatum, whereas neuropeptide Y-immunoreactive neurons were preferentially found in the ventromedial areas of this structure. Electron microscopic observations on sections processed under either condition revealed that choline acetyltransferase-positive terminals form synaptic contacts of the symmetrical type with neuropeptide Y-positive somata and proximal dendrites and that choline acetyltransferase-positive neurons are contacted by neuropeptide Y-positive terminals. These data show that the striatal neuropeptide Y- and choline acetyltransferase-containing neuronal systems have reciprocal synaptic interactions and provide morphological support for the hypothesis that striatal cholinergic and neuropeptide Y interneuron activities may be functionally linked.

Ultrastructural features of the choline acetyltransferase-containing neurons and relationships with nigral dopaminergic and cortical afferent pathways in the rat striatum.

The aim of this study was first to specify the morphology and neuronal environment of the large cholinergic neurons, and second to determine the distribution and mode of termination of the corticostriatal and dopaminergic inputs on these neurons in the rat striatum. Immunocytochemical procedures with a monoclonal antibody against choline acetyltransferase, Golgi staining and standard electron microscopic techniques were used to specify the ultrastructural features of the putatively cholinergic classical large neurons. The large/choline acetyltransferase-positive neurons are characterized by a voluminous, eccentric, and deeply indented nucleus leaving a large cytoplasmic area, and by the presence of an abundant granular endoplasmic reticulum and of many polysomes and free ribosomes. Serial ultrathin sectioning further indicated the presence of nematosomes or nucleolus-like bodies within the nucleus and the cytoplasm of the large neurons. In addition, these neurons were found to be in direct apposition with up to four surrounding neurons showing features typical of medium-sized spiny neurons. These data support the view that the putatively cholinergic neurons may have an intense metabolic activity and may be involved in striatal clusters. When choline acetyltransferase immunostaining was coupled with the identification of degenerating corticostriatal afferents after lesion of the cerebral cortex, degenerating terminals were seen to form synapses of an asymmetrical type on distal labelled dendrites, but these contacts were very rare. On the other hand, nigrostriatal dopaminergic axons, identified by means of either the degeneration method or tyrosine hydroxylase immunostaining, were often found to run directly for long distances around the choline acetyltransferase-positive cell bodies. Occasionally, dopaminergic terminals formed possible symmetrical synapses on choline acetyltransferase-positive cell bodies or proximal dendrites. These data provide evidence that the putatively cholinergic neurons are directly contacted by corticostriatal and dopaminergic nigrostriatal afferents. The respective positions and nature of the two types of contacts further provide morphological support for the hypothesis that postsynaptic interactions may occur between the corticostriatal and dopaminergic nigrostriatal afferents at the level of the cholinergic neurons.

Emergence of a synaptic neuronal network within primary striatal cultures seeded in serum-free medium.

In order to investigate the basic cellular mechanisms involved in neuronal interactions within the striatum, we prepared a primary striatal cell culture from rat fetal brain in chemically defined medium. Using morphological and whole-cell recording methods, we observed that an intensive neuritic elongation with a progressive build up of a sodium-dependent electrogenesis occurred during the first week of culture. Morphologically mature synapses began to develop after 10 days in vitro. By this time, most of the neurons (82 +/- 9%) received spontaneously synaptic potentials, which led them to fire (71 +/- 11%). The spontaneous firing was prevented by cadmium (200 microM) and tetrodotoxin (5 microM), which suggested that a Ca(2+)-dependent release of neurotransmitters was involved in the synaptic activation. We further obtained evidence that GABA, and to a lesser extent acetylcholine, contributed to these spontaneous synaptic potentials. At 15 days in vitro, it was possible to observe up to four synaptic contacts on a given dendrite. By this time, whole-cell recordings performed on pairs of neurons showed that the mature neurons were interconnected by excitatory synapses. As the number of synapses increased, the striatal neurons gradually formed a large network in which spontaneous activity developed, which tended to be organized into synchronized bursting patterns.