Ropinirole versus L-DOPA effects on striatal opioid peptide precursors in a rodent model of Parkinson's disease: implications for dyskinesia

The dopamine precursor, L-3,4-dihydroxyphenylalanine (L-DOPA), remains the most common treatment for Parkinson's disease. However, following long-term treatment, disabling side effects, particularly L-DOPA-induced dyskinesias, are encountered. Conversely, D2/D3 dopamine receptor agonists, such as ropinirole, exert an anti-parkinsonian effect while eliciting less dyskinesia when administered de novo in Parkinson's disease patients. Parkinson's disease and L-DOPA-induced dyskinesia are both associated with changes in mRNA and peptide levels of the opioid peptide precursors preproenkephalin-A (PPE-A) and preproenkephalin-B (PPE-B). Furthermore, a potential role of abnormal opioid peptide transmission in dyskinesia is suggested due to the ability of opioid receptor antagonists to reduce the L-DOPA-induced dyskinesia in animal models of Parkinson's disease. In this study, the behavioural response, striatal topography and levels of expression of the opioid peptide precursors PPE-A and PPE-B were assessed, following repeated vehicle, ropinirole, or L-DOPA administration in the 6-OHDA-lesioned rat model of Parkinson's disease. While repeated administration of L-DOPA significantly elevated PPE-B mRNA levels (313% cf. vehicle, 6-OHDA-lesioned rostral striatum; 189% cf. vehicle, 6-OHDA-lesioned caudal striatum) in the unilaterally 6-OHDA-lesioned rat model of Parkinson's disease, ropinirole did not. These data and previous studies suggest the involvement of enhanced opioid transmission in L-DOPA-induced dyskinesia and that part of the reason why D2/D3 dopamine receptor agonists have a reduced propensity to elicit dyskinesia may reside in their reduced ability to elevate opioid transmission.     

Further studies on possible dynorphin involvement in the ovulatory luteinizing hormone surge in the proestrous rat

A decrease in inhibitory tone of endogenous opioid peptide on the afternoon of proestrus is one event underlying generation of the ovulatory luteinizing hormone (LH) surge, since premature removal of this inhibitory tone (i.e., disinhibition) results in an early onset of the surge. Our laboratory demonstrated that blockade of κ-opioid receptors in the medial preoptic area (MPOA) advanced the onset of the LH surge on proestrus. Since dynorphin is the endogenous ligand for the κ-opioid receptor, the present studies examined the possible role of dynorphin in this disinhibition response. 1) Neutralization of endogenous dynorphin peptides, by push-pull perfusion of the MPOA with antibodies specific for dynorphin A_1–17 or A_1–8 from 1030–1355 h on proestrus, tended to prematurely advance the increase in plasma LH levels normally occurring on this day of the estrous cycle. Although this increase was not statistically significant when compared with controls, plasma LH levels in two antibody-treated rats were sufficiently elevated to cause full ovulation, a response that did not occur in controls. These data suggest that dynorphin A_1–17 and A_1–8 might have a role in the MPOA, although a minor one, in suppressing LH secretion early on proestrus. MPOA levels of prodynorphin mRNA decreased at 1700–1800 h on proestrus when plasma LH levels were high, compared with values at 1300–1400 h when plasma LH levels were low. This change did not occur on diestrous d 1 when there was no LH surge. 2) MPOA levels of κ-opioid receptor mRNA did not change on proestrus or diestrous d 1. These results suggest that a reduction in prodynorphin gene expression on the afternoon of proestrus may be one event involved in a possible decrease in dynorphin inhibitory tone on the ovulatory LH surge-generating signal.     

Dynorphin-A immunoreactive terminals on the neuronal somata of rat mesencephalic trigeminal nucleus

Dynorphin-A-like immunoreactivity was investigated in the rat mesencephalic trigeminal nucleus (Mes 5) at the light and electron microscopic levels. Dynorphin-A immunoreactive fibers and puncta, likely representing nerve terminals, were observed throughout rostrocaudal extension of the Mes 5 at the light microscopic level. Within the rostrocaudal extension, more abundant fibers and puncta were localized in the midbrain–pontine junction and pontine areas than in the midbrain area. At the electron microscopic level, dynorphin-A immunoreactive synapses were observed on the somata of Mes 5. Dynorphin-A-like immunoreactivity tended to be restricted to dense-cored vesicles in the synapses. These results suggest that dynorphin-A-containing fiber systems affect mastication through the Mes 5.     

Striato-nigral dynorphin and substance P pathways in the rat

Summary In the present study the functional role of the striato-nigral dynorphin and substance P pathways in rat brain has been studied using the rotational behavioural model and an intracerebral dialysis technique complemented with brain lesions and immunohistochemical analysis. Attempts were made to evaluate whether these striato-nigral neurons have a feed-back modulatory action on the dopaminergic nigro-striatal system, or whether they represent an outflow pathway conveying motor information from the striatum. Unilateral injection of dynorphin A into the substantia nigra reticulata of naive rats induced contralateral rotational behaviour. This effect was dose-dependent and mimicked by the kappa-opioid receptor agonist, U50,488H. Intranigral injection of substance P, as well as substance K, also produced dose-dependent contralateral rotational behaviour. Unilateral injections of ibotenic acid into various sites of the striatum were used to destroy the striato-nigral pathways. The lesions produced a depletion of dynorphin- and substance P-like immunoreactivity in the pars reticulata of the substantia nigra ipsilateral to the lesion and markedly affected the behavioural responses to intranigral peptide injections. Dynorphin A more potently induced contralateral rotation in the lesioned compared to naive non-lesioned rats, suggesting development of supersensitivity for this peptide. Substance P on the other hand, was markedly less potent in inducing rotation in lesioned animals. The rotational responses to both dynorphin A and substance P were potentiated by injection of amphetamine 1 h later, suggesting that both peptides act via nigro-striatal dopamine neurons. However, in rats with unilateral nigro-striatal dopamine denervation, produced with 6-hydroxy-dopamine, dynorphin A retained its potency to induce rotational behaviour; substance P was again much less potent. Thus, both the ibotenic acid and 6-hydroxy-dopamine lesions differently affect the action of dynorphin A and substance P in the zona reticulata of the substantia nigra. The data suggests that substance P requires an intact dopamine pathway to produce the rotational response, while dynorphin A does not. Direct evidence that behavioural activation produced by dynorphin A is not dependent upon dopamine stimulation was obtained by intrastriatal dialysis experiments in which changes in striatal dopamine release were measured following intranigral injection of dynorphin A or substance P. Intranigral dynorphin A in fact reduced, while substance P increased the release of dopamine. It is concluded that the dynorphin and substance P striatonigral pathways have different functions. Thus, substance P in the striato-nigral pathway may have a role in a positive feed-back loop regulating the firing of nigro-striatal dopamine neurons, while dynorphin might be important in negative feed-back control. The rotational behaviour produced by DYN A is probably due to direct stimulation of receptors located on nigro-thalamic and nigro-tectal pathways.     

Variation in hippocampal dynorphin b-immunoreactive mossy fiber terminal fields of apomorphine-(un)susceptible rats

The size of distinct hippocampal sub-fields were measured in the apomorphine-susceptible and apomorphine-unsusceptible rat lines. Mossy fiber terminal fields were delineated using dynorphin B immunoreactivity and area measurements were taken from (1) the supra-pyramidal mossy fiber terminal field; (2) the intra- and infra-pyramidal mossy fiber terminal field; (3) the hilus of the fascia dentata (4) the non dynorphin B immunoreactive area of the regio inferior and fascia dentata and (5) the total area of regio inferior and fascia dentata. The data indicate that statistically significant differences in the morphometry of the hippocampal subfields of the apomorphine susceptible and unsusceptible rats are confined to the intra- and infra terminal field: the relative size of the left and right intra- and infra terminal field of apomorphine unsusceptible rats are significantly larger than those of the apomorphine susceptible rats. These data explain at least in part the differential response of these rats to novelty.     

Striatal and ventral pallidum dynorphin concentrations are markedly increased in human chronic cocaine users

Interest in development of therapeutics targeting brain neuropeptide systems for treatment of cocaine addiction (e.g., kappa opioid agonists) is based on animal data showing interactions between the neuropeptides, brain dopamine, and cocaine. In this autopsied brain study, our major objective was to establish by radioimmunoassay whether levels of dynorphin and other neuropeptides (e.g., metenkephalin, neurotensin and substance P) are increased in the dopamine-rich caudate, putamen, and nucleus accumbens of human chronic cocaine users (n=12) vs. matched control subjects (n=17) as predicted by animal findings. Changes were limited to markedly increased dynorphin immunoreactivity in caudate (+92%), decreased caudate neurotensin (-49%), and a trend for increased dynorphin (+75%) in putamen. In other examined subcortical/cerebral cortical areas dynorphin levels were normal with the striking exception of the ventral pallidum (+346%), whereas cerebral cortical metenkephalin levels were generally decreased and neurotensin variably changed. Our finding that, in contradistinction to animal data, the other striatal neuropeptides were not increased in human cocaine users could be explained by differences in pattern and contingency between human drug users and the animal models. However, the human dynorphin observations parallel well animal findings and suggest that the dynorphin system is upregulated, manifested as elevated neuropeptide levels, after chronic drug exposure in striatum and ventral pallidum. Our postmortem brain data suggest involvement of striatal dynorphin systems in human cocaine users and should add to the interest in the testing of new dynorphin-related therapeutics for the treatment of cocaine addiction.     

携带前强啡肽基因的Ad5 F35重组腺病毒制备及鉴定

目的 用Ad5F35载体制备携带前强啡肽基因的重组腺病毒Ad5F35-PDP并对其进行鉴定.方法 利用NheI和Agel分别对合成的目的 基因prodynorphin聚合酶链反应(PCR)产物和pDC316-LacZ-a载体质粒进行双酶切.将PDP目的 基因片段和线性化的pDC316连接,克隆构建pDC316-PDP.pDC316-PDP与腺病毒骨架pBHG-fiber5/35共转染293细胞制备Ad5F35-PDP重组腺病毒,收获并纯化病毒,检测重组病毒滴度,运用PCR进行鉴定.结果 编码PDP的基因序列经测序鉴定证实与Gene Bank中的一致,pDC316-PDP构建成功.pDC316-PDP与腺病毒骨架共转染293细胞后见明显的毒斑,说明两者在293细胞中同源重组并包装成功.经PCR证实Ad5F35-PDP重组腺病毒构建完成.病毒滴度为1×1012v.P./ml.结论 本实验成功制备了含PDP全序列的高滴度、高纯度的Ad5F35-PDP重组腺病毒,为下一步进行该重组病毒生物安全性能和生物学功能研究及今后应用Ad5F35-PDP重组腺病毒对吗啡成瘾患者进行基因治疗奠定了基础.     

Levels of prodynorphin mRNA in rat dentate gyrus are decreased during hippocampal kindling

The effect of hippocampal kindling on the levels of prodynorphin mRNA in rat hippocampus was examined by in situ hybridization using a synthetic oligonucleotide probe. Cryostat tissue sections were hybridised with a ³²P-labelled 100 mer DNA probe complementary to the coding region of rat prodynorphin mRNA, and exposed to X-ray film. In rats exhibiting stage 4 seizures, the levels of prodynorphin mRNA in the dentate gyrus were dramatically reduced compared to control animals. This suggests that the development of kindling is accompanied by a reduction in the rate of synthesis of peptides derived from prodynorphin.