重复注射6-羟多巴胺建立帕金森病动物模型

目的探讨通过大鼠中脑内重复注射 6-羟多巴胺 (6-hydroxydopamine,6-OHDA)建立高效、稳定、可靠的帕金森病动物模型。方法将 60 只雄性 SD 大鼠随机分为一次打击组和二次打击组,经腹腔注射 4%水合氯醛(40 mg / 100g)麻醉,脑立体定位仪固定,于左侧黑质致密部(SNC)和中脑腹侧被盖区(VTA)分别注射 6-羟多巴胺 (6-OHDA,2 g / L,2μl),二次打击组一周后在相同位置重复注射同剂量的 6-OHDA,建立帕金森模型,观察其行为改变,通过 HE、TH 和 DIG-dUTP 染色观察其细胞形态的改变及凋亡情况。结果经过二次打击的大鼠,模型成功率(旋转周数 >7 r / min)为 86.7%,明显多于一次打击组的 33.3%;HE 染色显示,二次打击组凋亡细胞的阳性率(37.12%)明显多于一次打击组(21.25%);DIG-dUTP 染色显示,一次打击组大鼠左侧中脑黑质区神经细胞肿胀的数量多 ,凋亡数量少,凋亡细胞阳性率为 20.73%,二次打击组细胞凋亡数显著增多,凋亡细胞阳性率达 36.03%;TH 染色显示,二次打击组 TH 阳性细胞数明显减少,TH 细胞阳性率(18.61%)显著低于一次打击组(36.55%)。结论通过二次打击建立帕金森病动物模型成功率高于一次打击。     

Interaction between noradrenergic and cholinergic systems in the rat brain: behavioural function in locomotor activity.

Anticholinergic drugs such as scopolamine and atropine induce a mild locomotor stimulation when given intraperitoneally to rats. This effect is usually ascribed to interaction between dopaminergic and cholinergic transmission in the striatum or nucleus accumbens. However, an interaction of acetylcholine with noradrenergic systems is also apparent from biochemical data and the results reported here indicate that at least part of the locomotor activity induced by scopolamine or atropine involves a noradrenergic component. Depletion of forebrain noradrenaline by injection of 4 μg of the selective neurotoxin 6-hydroxydopamine into the dorsal bundle was found to potentiate the locomotor activation induced by various doses of scopolamine or atropine. This was a central effect since methylscopolamine, which does not pass the blood-brain barrier, failed to induce locomotor activity and was not affected by the noradrenergic lesion. The noradrenergic interaction was restricted to cholinergic drugs since locomotor activity induced by the indirect dopamine agonist amphetamine was not affected by noradrenaline depletion.These studies show that the interaction between noradrenergic and cholinergic transmission, which has previously been indicated by biochemical analysis, influences behaviour and they also cast some light on the functions of the central noradrenergic system itself.     

Influence of 6-hydroxydopamine and of α-methyl-p-tyrosine on the effects of some centrally acting agents

The central effects of morphine, cocaine, phenidylate and chlorpromazine were studied in mice treated with intracerebral injections of 6-hydroxydopamine (6-OHDA). The response to morphine and phenidylate of the animals pretreated with 6-OHDA consisted only of local stereotypies, while the response to cocaine was almost completely blocked. Mice receiving 250 mg/kg i.p. of α-methyl-p-tyrosine (αMT) reacted to morphine and amphetamine only with a very slight increase of motor activity. The excitation induced in cats by morphine was prevented by previous treatment with αMT. The modifications of the responses to the various centrally acting compounds induced by 6-OHDA and αMT are discussed in terms of the depleting effects of the drugs on brain catecholamines.     

Reciprocal relations between monoaminergic brain systems as a factor in pharmacological compensation of behavioral disturbances induced in animals by 6-hydroxydopamine

Institute of Biological Physics. Academy of Sciences of the USSR, Pushchino. (Presented by Academician of the Academy of Medical Sciences of the USSR A. V. Val'dman.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 1, pp. 52–54, January, 1989.     

Feeding induced in cats by electrical stimulation of the brain stem

Summary Stimulation at 14 of 106 loci (Figs. 1 and 2) at which electrodes had been chronically implanted elicited immediate feeding in fully satiated, freely moving cats. Half of the effective points were in the lateral hypothalamus and thus agreed with expectations from extensive prior research on the neural organization of feeding behavior. The other points, however, were in the ventral tegmental area or at the ventrolateral boundary of the central gray. The distribution of all effective points could be explained as following a projection pathway from the globus pallidus. Exploration along 162 recording tracks in acute experiments on these same cats revealed no consistent differences in projection of evoked potentials which could distinguish stimulation at effective versus ineffective loci. Potentials of possible significance, however, were evoked in the paramedial nucleus reticularis of the medulla, the rostral pontine nuclei, the nucleus centralis superior of Bekterev, the lateral frontal cortex and the basal medial forebrain in the vicinity of the diagonal band of Broca.Supported by USPHS Grant B 1068. Dr. Wyrwicka held a fellowship from Foundations' Fund for Research in Psychiatry. The authors are indebted to Mr. Joseph Jones and Mrs. Frances Bignall for technical assistance.     

Intrapallidal injection of 6-hydroxydopamine induced changes in dopamine innervation and neuronal activity of globus pallidus

The globus pallidus (GP) plays an important role in basal ganglia circuitry. In contrast to the well-characterized actions of dopamine on striatal neurons, the functional role of the dopamine innervation of GP is still not clearly determined. The present study aimed to investigate the effects of intrapallidal injection of 6-hydroxydopamine (6-OHDA) on rotational behavior induced by apomorphine, on the loss of dopamine cell bodies in the substantia nigra pars compacta (SNc) and fibers in the GP and striatum and on in vivo extracellularly-recorded GP neurons in the rat. Injection of 6-OHDA into GP induced severe loss of tyrosine hydroxylase–immunoreactive (TH-IR) fibers in GP (−85%) with a reduction in the number of TH-IR cell bodies in the SNc (−52%) and fibers in the striatum (−50%). S.c. injection of apomorphine in these rats induced a moderate number of turns (26±6 turns/5 min). Electrophysiological recordings show that 6-OHDA injection in GP induced a significant decrease of the firing rate of GP neurons (16.02±1.11 versus 24.14±1.58 spikes/sec in control animals and 22.83±1.28 in sham animals, one-way ANOVA, P<0.0001) without any change in the firing pattern (χ²=1.03, df=4, P=0.90). Our results support the premise of the existence of collaterals of SNc dopaminergic axons projecting to the striatum and GP and that dopamine plays a role in the modulation of the firing rate but not the firing pattern of GP neurons. Our data provide important insights into the functional role of the SNc–GP dopaminergic pathway suggesting that dopamine depletion in GP may participate in the development of motor disabilities.     

Effects of graft pooling of foetal rat and mouse tissue and immunosuppression in the 6-hydroxydopamine rat model of Parkinson’s disease

 We employed intracerebral co-transplantation of foetal xenogeneic striatal mouse tissue and allogeneic rat substantia nigra into the adult rat brain to elucidate the effects of xenogeneic mouse graft on the function and survival of an allogeneic rat graft in 6-hydroxydopamine lesioned Sprague-Dawley rats. Foetal mouse striatum (STR) and rat substantia nigra (VM) were transplanted as non-pooled separate deposits or a pooled cell suspension with or without cyclosporin A (Cy A). Immunosuppressed recipients of pooled rat and mouse co-grafts showed a significantly better compensation of amphetamine-induced rotational behaviour compared with non-immunosuppressed animals with pooled rat and mouse co-grafts 3 and 6 weeks post-grafting.Tyrosine hydroxylase (TH) immunohistochemistry revealed a non-significant reduction in survival in pooled (1806.3±367.5 cells) rat and mouse co-transplants without immunosuppression compared with immunosuppressed pooled (3383.3±732.7 cells) animals with allo- and xenogeneic tissue and controls (3506.4±839.3 cells). Graft volumes were significantly reduced in pooled transplants without immunosuppression (0.1±0.026 mm³; ANOVA post-hoc SchefféF-test, P<0.0001) compared with immunosuppressed recipients (0.7±0.1 mm³) and controls (0.6±0.1 mm³). In non-pooled allo- and xenogeneic grafts without immunosuppression the survival rate of the TH-immunoreactive cells and graft volumes were reduced (2359.3±479.5 cells; 0.2±0.043 mm³) compared with immunosuppressed animals (2927.3±946.6 cells; 0.6±0.2 mm³) and controls (2701.1±693.8 cells; 0.3±0.1 mm³) without reaching a level of significance. Rejection of mouse tissue was observed in all non-immunosuppressed recipients. In summary: (i) continued immunosuppression yielded significant beneficial effects on function and beneficial effects on survival of pooled grafts with an immunogenetic disparity; (ii) the rejection of a xenogeneic graft component may compromise survival and function of other, allogeneic graft components; and (iii) transplantation of non-pooled allo- and xenogeneic tissues may result in a better survival of the graft compared with pooled cell suspensions. Received: 25 March 1996 / Accepted: 1 December 1996     

GM1 ganglioside enhances regrowth of noradrenaline nerve terminals in rat cerebral cortex lesioned by the neurotoxin 6-hydroxydopamine

The effect of exogenous GM1 ganglioside on selectively noradrenaline-denervated rat cerebral cortex was investigated by measuring the spatial distribution of endogenous noradrenaline levels and by fluorescence histochemical analysis. A local noradrenaline denervation was produced by intracortical infusion of the selective catecholamine neurotoxin 6-hydroxydopamine for 3 or 7 days. The neurotoxin infusion caused an almost complete noradrenaline denervation in a restricted area around the infusion point as reflected by an almost complete long-term disappearance of noradrenaline nerve terminals and reduction of noradrenaline levels. There was with time a slow recovery of the levels, most likely related to a spontaneous noradrenaline nerve terminal regeneration. Post-treatment for 1 week with GM1 had very small effects on the 6-hydroxydopamine-induced reduction of the noradrenaline levels, while pretreatment with GM1 for 3 days before the neurotoxin infusion and continuing the GM1 administration for another 7-14 days significantly enhanced noradrenaline recovery, as observed both bio- and histochemically. GM1 had no effect on the 6-hydroxydopamine-induced noradrenaline depletion acutely, indicating that GM1 does not interfere with the direct neurotoxic actions of 6-hydroxydopamine. The present results thus indicate that exogenous GM1 enhances regrowth of noradrenaline nerve terminals which may be due to a regrowth stimulatory effect (regeneration/collateral sprouting) and/or related to protective actions of GM1 against retrograde degeneration of noradrenaline axons following the neurotoxin-induced lesion.     

Collateral sprouting and reduced activity of the rat mesocortical dopaminergic neurons after selective destruction of the ascending noradrenergic bundles

The properties of the mesocortical dopaminergic neurons projecting to the pregenual and anterior supragenual cortices were examined 3–6 months after the degeneration of ascending noradrenergic pathways caused by bilateral multiple or single microinjections of 6-hydroxydopamine made laterally to the pedunculus cerebellaris superior. In all rats and in all cortical areas examined, noradrenaline levels were reduced by more than 75%. A similar decrease in noradrenaline levels was obtained in the ventral tegmental area. As indicated by the increases in cortical levels of dopamine and in [³H]dopamine specific uptake sites as well as by histochemical analysis, these lesions induced a collateral sprouting of the mesocortical dopaminergic neurons. The intensity of the effect varied from one animal to another and even from one anteromedial hemicortex to another. When present, the increase in dopamine levels was observed in all the cortical areas investigated. As suggested by the decreased ratio of the amount of dihydroxyphenylacetic acid to dopamine in the cortex, the activity of the mesocortical dopaminergic neurons was reduced in the rats with lesions. This effect was even seen in rats in which the cortical levels of dopamine were only slightly increased. Both the collateral sprouting and the reduced activity of the mesocortical dopaminergic neurons were related to the degeneration of the noradrenergic neurons and not to a non-specific effect of 6-hydroxydopamine, since both phenomena did not occur in rats pretreated with desipramine, a treatment which prevented the decline in noradrenaline levels.Thus, a lesion of the ascending noradrenergic pathways can lead to sprouting of dopaminergic neurons in the cortex and a reduced activity of these dopaminergic neurons. The respective role of the disappearance of the noradrenergic innervation in the cerebral cortex and in the ventral tegmental area in the collateral sprouting and in the reduced activity of the mesocortical dopaminergic neurons is discussed.     

Development of the visual cortex of rats treated with 6-hydroxydopamine in early life

Summary The development of the visual cortex was examined in control rats and in littermates whose cortical noradrenergic innervation was removed with 6-hydroxydopamine. Qualitative and quantitative observations revealed no differences in the lamination, cytoarchitecture, cell density and size between the two groups of animals. These results suggest that the number, size, and distribution of cortical neurons are not significantly altered in the absence of noradrenergic afferents.     

Apomorphine-induced restoration of drinking to thirst challenges in 6-hydroxydopamine-treated rats.

After bilateral injections of 6-hydroxydopamine along the ascending DA fibers or after lateral hypothalamic electrocoagulation, rats become aphagic and adipsic, and do not drink in response to hypertonic saline or isoproterenol. However, after low doses of the DA-receptor stimulating agent, apomorphine, 6-OH-DA lesioned rats drink near-normal quantities of water in a 30 min test following either regulatory challenge. Apomorphine does not restore drinking to these thirst stimuli in rats with lateral hypothalamic electrocoagulations. These findings (1) provide additional evidence that the ingestive impairments seen after 6-OH-DA injections are due to degeneration of DA-containing neurons, and (2) suggest that the lateral hypothalamic electrocoagulation is functionally different from a specific lesion of DA neurons, possibly by interrupting striatal efferent pathways in addition to the DA afferent fibers. The interruption of these striatal efferents may explain why apomorphine does not restore drinking in the animal with lateral hypothalamic lesions.     

Stimulation of resistance to 6-hydroxydopamine in a human neuroblastoma cell line by nerve growth factor

Resistance of human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) to the neurotoxin 6-hydroxydopamine (6-OHDA) was established by exposure of the cells to nerve growth factor (NGF). SY5Y cells display several properties of immature sympathetic nerve cells, including morphological responses to nerve growth factor and susceptibility to cytolysis by 6-OHDA. High resistance to 6-OHDA was achieved by culturing SY5Y cells with NGF. Protection persisted when NGF was absent for 24 h or when cells were treated with colcemid. In contrast, dibutyryl cyclic AMP did not stimulate resistance to 6-OHDA. NGF-treated cells were also resistant to H2O2, a toxic product of 6-OHDA autoxidation.     

Increased β1-adrenoceptor density after 6-hydroxydopamine pretreatment in rat colon and lung

Estimation of β-adrenoceptor-binding sites with ¹²⁵I-(-)-pindolol in rat colon show a proportion of 30%β₁,-adrenoceptors and 70%β₂-adrenoceptors. Studies on the isolated colon strip have revealed a neuronal β-adrenoceptor involved in the inhibitory response of colon motility to β-adrenoceptor stimulation. In order to further characterize the β-adrenoceptors in the colon, acute and chronic treatments with 6-hydroxydopamine were made. Both acute pretreatment of rats with 6-hydroxydopamine for 8 and 24 h (one intravenous injection) and chronic treatment for 3 days (implanted osmotic mini-pumps), reduced the noradrenaline tissue content by 90%, and successively increased the β-adrenoceptor-binding sites from 14.3 to 21.7 fmol mg^_1 P^_1 in colon and from 158 to 240 fmol mg^-1 P^_1 in lung membranes. Displacement of the radiolabelled ligand by the selective β-adrenoceptor antagonists, pafenolol and ICI 118.551 showed that the density of β₁,-adrenoceptor binding sites was more than doubled, whereas the density of β₂-adrenoceptor-binding sites was only marginally increased by chronic treatment with 6-hydroxydopamine. Thus sympathetic denervation by 6-hydroxydopamine treatment produced a selective increase in β₁-adrenoceptors in the rat colon. These results may indicate that stimulation of β₁-adrenoceptors in both colon and lung have a neuronal linkage.     

Visual discrimination learning and interhemispheric transfer in the cat,as affected by 6-hydroxydopamine

Summary Learning and interhemispheric transfer of visual flux, pattern and form discriminations were studied in the cat after selected exposure of one suprasylvian cortex to 6-hydroxydopamine (6-OHDA). Biochemical assay using High Performance Liquid Chromatography (HPLC) two weeks after 6-OHDA revealed no discernible norepinephrine or dopamine in the treated cortex, but elevated concentrations of these transmitters in the cortex of the opposite hemisphere. Visual discriminations learned before treatment with 6-OHDA were retained at a high level using either the eye on the side of chemical lesion or the eye on the untreated side. An asymmetric deficit in learning new form discriminations was present, however, when the eye on the untreated side was used, in contrast to normal learning using the eye on the side of the hemisphere with depleted adrenergic nerve supply. Once learning was achieved using the lesioned hemisphere transfer of the engram was found to the untreated hemisphere. Thus, the unlesioned hemisphere was unable to learn normally using direct retinal input from the ipsilateral eye, but showed good capacity for learning using indirect visual input from the contralateral eye. This suggests a powerful influence of the callosum on the learning abilities of the two hemispheres, an influence proved by sectioning the callosum. Callosotomy resulted in a reversal of the discriminative capacities seen after 6-OHDA, i.e. the lesioned hemisphere was defective relative to the unlesioned hemisphere.This research was supported in part by USPHS research grant EY-00577, EY-01583 and SO7-RR05415     

Substance P modifies the 6-hydroxydopamine induced alteration of postnatal development of central noradrenaline neurons

Systemic treatment of new-born rats with the catecholamine neurotoxin 6-hydroxydopamine leads to a permanent and selective alteration of the postnatal development of the central noradrenaline neurons, in particular of the locus coeruleus system. The changes involve a pronounced and permanent degeneration of distant nerve terminal projections (e.g. in the cerebral cortex and spinal cord) and a hyperinnervation of regions close to the noradrenaline perikarya (e.g. in the cerebellum and pons-medulla). Substance P administered intracisternally was found to counteract significantly both the 6-hydroxydopamine-induced denervation and hyperinnervation, as monitored by measuring endogenous noradrenaline levels and [3H]noradrenaline uptake in vitro. The counteracting effect of substance P disclosed a clear dose-response relationship and was most effective when injected on postnatal days one and two, while practically no effects were observed after injection on postnatal days three and four. Substance P treatment alone of new-born rats had no effect on the postnatal development of the regional monoamine levels. Binding studies employing radioligand technique showed substance P treatment to abolish the 6-hydroxydopamine-induced increase in beta-receptor binding in the frontal cortex, suggesting the 'spared' noradrenaline terminals to be functionally active. Substance P was shown to increase the utilization of noradrenaline in the neonatal stage. The results indicate that the counteracting effect of substance P may be due to a prevention of degeneration, growth stimulation and/or trophic influences on central noradrenaline neurons, possibly related to an excitatory effect of substance P on noradrenaline neurons.     

Substantial reduction of noradrenaline in kitten visual cortex by intraventricular injections of 6-hydroxydopamine does not always prevent ocular dominance shifts after monocular deprivation

Summary Ten kittens had cannulas inserted into their lateral ventricles for daily injections of 6-hydroxydopamine (6-OHDA). At 5–6 weeks of age one eye was sutured shut, and one week later recordings were made from the visual cortex to assay the ocular dominance of a sample of cells. In six kittens the injections of 6-OHDA were continued until the day before recording, while in four kittens the injections were stopped around the time of eye suture, on the assumption that continued injections of 6-OHDA over several days has effects that are not specific to the noradrenaline (NA) system and that the two procedures might show different results. In all animals the concentration of NA in the visual cortex near the site of recording was reduced by approximately 90%. In all animals the ocular dominance histograms recorded from the visual cortex were shifted so that the majority of cells (83 ± 13%) were dominated by the open eye. There were no substantial differences between the two groups of experimental animals or between the experimental animals and two control animals that had cannulas implanted and ascorbate alone injected without 6-OHDA. We conclude that the concentration of NA in the visual cortex can be reduced substantially by injections of 6-OHDA into the lateral ventricle without preventing the shift in ocular dominance that usually occurs after suturing shut the eyelids of one eye.     

Impaired growth of the cerebral cortex of rats treated neonatally with 6-hydroxydopamine under different environmental conditions

Male Wistar rat pups received subcutaneous injections of either 100 mg/kg 6-hydroxydopamine (6-OHDA) or saline on days 1, 3, 5 and 7. The noradrenaline (NA) content of the cerebral cortex was reduced by 70% while sleep registration during the first two weeks of life did not show any significant differences between drug- and saline-treated animals. After weaning (day 25), both 6-OHDA- and saline-injected animals were reared under standard and enriched environmental conditions. Whereas a clear increase in cerebral cortical weight after experience with an enriched environment was found in saline-treated rats, 6-OHDA-treated animals had lower cortical weights and showed less increase due to the environment. These results demonstrate that catecholamine neurotransmission during early development influences the development and plasticity of the cerebral cortex.     

Acute immediate-early gene response to 6-hydroxydopamine infusions into the medial forebrain bundle

Although the long-term neurobiological and behavioral effects of nigrostriatal lesions are well characterized, the events occurring soon after injury are not. These acute events can provide insight into the mechanisms underlying long-term adaptations to nigrostriatal lesions. The present experiments examined the basal ganglia immediate-early gene response to infusions of the catecholamine neurotoxin 6-hydroxydopamine into the nigrostriatal pathway in rats. Following 6-hydroxydopamine infusions into the medial forebrain bundle in awake, behaving rats, there was a rapid and transient induction of striatal c-fos and zif/268 messenger RNAs. Both immediate-early genes were maximally induced by 45min post-infusion, and returned to control levels by 1.5h (c-fos) or 3h (zif/268) post-infusion. Double-labeling experiments revealed that striatal c-fos expression occurred preferentially in preproenkephalin-expressing neurons. 6-Hydroxydopamine-induced c-fos messenger RNA was also observed in the substantia nigra pars reticulata and entopeduncular nucleus, but not the globus pallidus, 45 min after medial forebrain bundle 6-hydroxydopamine infusions. Finally, the role of ionotropic striatal glutamate receptors in nigrostriatal injury-induced striatal c-fos was examined by combining medial forebrain bundle 6-hydroxydopamine infusions with intrastriatal glutamate antagonist infusions. Both the N-methyl-D-aspartate antagonist, (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid, and the non-N-methyl-D-aspartate antagonist, 6,7-dinitroquinoxaline-2, 3-dione, blocked striatal induction of c-fos messenger RNA following 6-hydroxydopamine infusions into the medial forebrain bundle.These results provide evidence of rapidly developing, glutamate-dependent molecular responses in the basal ganglia which may contribute to some of the well-described long-term adaptations of this system to nigrostriatal injury.     

Effects of intranasally applied dopamine on behavioral asymmetries in rats with unilateral 6-hydroxydopamine lesions of the nigro-striatal tract

Due to its lipophobic properties, dopamine is unable to cross the blood–brain barrier following systemic application. However, recently it has been demonstrated that, when applied directly via the nasal passages in the rat, dopamine exerts neurochemical and behavioural action, including increases of dopamine in striatal subregions, antidepressive-like action, and increased behavioral activity. These effects could potentially be mediated by exogenous dopamine acting as a direct agonist at postsynaptic dopamine receptors. However, it is also possible that intranasally applied dopamine acts indirectly via the modulation of the activity of dopaminergic cell bodies. To approach this question, the present study used rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal tract, as these lesions lead to pharmacologically stimulated behavioural asymmetries which are specific for direct and indirect dopamine agonists. We found that 7 days of repeated treatment with intranasal dopamine induced a sensitization of the turning response to amphetamine, but not to apomorphine. Furthermore, intranasal dopamine dose-dependently increased the use of the forepaw ipsilateral to the 6-OHDA-lesioned side of the brain. These results suggest that intranasally administered dopamine acts via an indirect mechanism of action, putatively by increasing the release of endogenous dopamine in the brain.