DA Fibers Research Articles

Therapeutic effects of fucoidan in 6-hydroxydopamine-lesioned rat model of Parkinson's disease: Role of NADPH oxidase-1.

To explore the effect of fucoidan treatment on oxidative stress-mediated dopaminergic neuronal damage and its potential mechanisms. The effect of fucoidan was investigated in a 6-hydroxydopamine (6-OHDA) rat model of PD, an animal model considered appropriate for preclinical studies of PD therapy. The effects of fucoidan treatment on animal behavior and the survival ratio of dopaminergic neurons were investigated. We further observed the effect of fucoidan on microglia and the NADPH oxidases-1 (Nox1), a family of enzymes generating reactive oxygen species (ROS). We found that chronic fucoidan administration mitigated the motor dysfunction induced by 6-OHDA. Similarly, fucoidan reduced the loss of DA neurons in the SNc and DA fibers in the striatum in 6-OHDA-lesioned rats. Moreover, we found that fucoidan inhibited the 6-OHDA-stimulating expression of Nox1 in both tyrosine hydroxylase (TH)-positive neurons and non-TH-positive neurons, prevented Nox1-sensitive oxidative stress and cell damage in SNc neurons. Fucoidan also effectively inhibited nigral microglial activation. These results support the beneficial effect of fucoidan in 6-OHDA-lesioned rat model of PD. Fucoidan may suppress the Nox1-triggered oxidative stress in the SNc to protect DA neurons from 6-OHDA-induced toxicity and achieve its beneficial effect.

Neuroprotective effects of the novel D 3/D 2 receptor agonist and antiparkinson agent, S32504, in vitro against 1-methyl-4-phenylpyridinium (MPP+) and in vivo against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): a comparison to ropinirole

The novel naphtoxazine derivative and preferential D 3 vs D 2 receptor agonist, S32504, restores perturbed motor function in rodent and primate models of antiparkinsonian activity with a potency superior to those of two further, preferential D 3 receptor agonists, pramipexole and ropinirole. However, potential neuroprotective properties of S32054 have not, to date, been evaluated. Herein, employing several measures of cellular integrity, we demonstrate that S32504 robustly, concentration-dependently and completely protects terminally differentiated SH-SY5Y cells against 1-methyl-4-phenylpyridinium (MPP+)-induced cell death in vitro. Further, S32504 was substantially more potent than pramipexole and ropinirole, the latter of which was neurotoxic at high concentrations. In vivo, subchronic treatment with low (0.25 mg/kg) and high (2.5 mg/kg) doses of S32504 prior to and during treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, MPTP, provided complete protection against MPTP-induced tyrosine hydroxylase immunoreactive (TH-IR) neuronal death in the substantia nigra pars compacta and ventral tegmental area. A high dose of ropinirole (2.5 mg/kg) provided some protection but statistical significance was not attained, and a low dose (0.25 mg/kg) was ineffective. Neither drug afforded protection against the MPTP-induced loss of DA fibers in the striatum, as measured by TH-IR and dopamine transporter immunoreactive fiber counts. In conclusion, the novel naphotoxazine and dopaminergic agonist, S32504, robustly protects dopaminergic neurones against the neurotoxic effects of MPP + and MPTP in in vitro and in vivo models, respectively. The underlying mechanisms and therapeutic pertinence of these actions will be of interest to further evaluate in view of its potent actions in behavioral models of antiparkinson activity.

Developing patterns of nitric oxide synthesizing neurons in the rat striatum: histochemical analysis

The prenatal and postnatal development of NADPH-diaphorase (NADPH-d)/neuronal nitric oxide synthase (nNOS) positive neurons was studied in the striatum of rats. NADPH-d was demonstrated enzyme histochemically and nNOS immunohistochemically using a polyclonal antibody. NADPH-d neurons appeared in the ventrolateral part of the striatum on embryonic day 18 (E18). Thereafter, the number of NADPH-d neurons increased and began to distribute homogeneously in the striatum. The density of NADPH-d neurons became highest at postnatal day 5 (P5) and then decreased as the volume of the striatum continued to increase. The number of NADPH-d neurons reached its peak around 3–4 weeks after birth. The sizes of NADPH-d neurons were measured. The NADPH-d neurons grew larger until P14 (mean area 260 μm 2) and became smaller thereafter (mean area 170 μm 2). Patches of high NADPH-d activity and tyrosine hydroxylase (TH) immunoreactivity were also examined in the developing striatum. The distributions of NADPH-d patches overlapped with those of TH-immunoreactive patches by P10. The spatiotemporal appearance of nNOS and overlapping of nNOS patchy distribution with TH point to an important role of NO and to an interaction between nNOS and DA fibers during development of the striatum.

Chapter 23 Vulnerability of monoaminergic neurons in the brainstem of the zitter rat in oxidative stress

In the monograph of Santiago Ramon y Cajal, he provided a detailed description about the morphological changes in degeneration and regeneration of peripheral and central nervous systems following lesions. He discussed factors that may promote or inhibit axonal growth after peripheral and/or central nerve injury. Cajal with a brilliant insight anticipated the existence of several factors acting on degeneration and regeneration. Free radicals have been proposed to be one of such factors. These highly reactive oxygen species-derived free radicals play a pathogenetic role in neurological disorders, including ischemia, trauma, Alzheimer's disease and Parkinson's disease (PD). In this review we will discuss the similarities and differences between the morphological changes under oxidant stress and Cajal's drawings of degeneration and regeneration following the central injury. The monoaminergic neuron systems in the brainstem appear vulnerable to these free radicals, which have also been implicated in the selective degeneration of the nigrostriatal DA system. We analyzed the degeneration of fibers and the neuronal cell death of brainstem monoaminergic neuron systems in a mutant rat, which has abnormal metabolism of oxygen species in the brain. The degeneration of DA cell bodies and fibers was characterized by swollen varicosities and clustered fibers.

Triple Colocalization of Tyrosine Hydroxylase, Calretinin, and Calbindin D-28k in the Periventricu lar-Hypophyseal Dopaminergic Neuronal System

Publisher Summary Studies have shown that the dopaminergic neurons terminating in the intermediate lobe of the pituitary (ILP) originate in the periventricular nucleus of the hypothalamus. A recent observation revealed that tyrosine hydroxylase (TH) colocalized with calretinin (CR), a calcium- binding protein, in the rat ILP. In addition, TH also colocalized with calbindin-D28k (CB), another calcium-binding protein (CaBP). The purpose of this chapter is to determine, using an immunohistochernical colocalization procedure, whether a triple colocalization of TH, CR, and CB existed in the DA fibers of the ILP. As it is already demonstrated that such a triple colocalization exists, the chapter sets out to map the dopaminergic cells in the rat hypothalamus in a search for the origin of these pituitary fibers. In immunocytochemical preparations of rat pituitary glands, varicose dopaminergic nerve fibers formed a plexus enveloping the endocrine cells of the intermediate lobe, and the lack of anti-DBH staining confirmed the DA nature of these fibers. Triple colocalization of TH, CR, and CB revealed that all three neurochemicals were present in these fibers in what appeared to be a 1:1 relationship between the CaBPs (CR, CB) and the TH-containing fibers. No immunoreactive cell bodies were observed in the ILP. Anatomical, neurochemical, and neuroendocrinological evidence indicates that dopamine terminals in the ILP originate from A14 cells in the hypothalamic periventricular nucleus. It has been observed that a triple colocalization of TH, CR, and CB was contained in about 3.3% of the A14 DA cell bodies, and it is suggested that these are the cells that innervate the ILP. Because of the triple colocalization within approximately 3.1% of the A11 cells, this system may also project to the ILP.

Synaptic and nonsynaptic monoaminergic neuron systems in the lamprey spinal cord.

In the lamprey spinal cord, dopamine- (DA) and 5-hydroxytryptamine-(5-HT) containing cells appear to play an important role in controlling the firing properties of motoneurons and interneurons and, thereby, in modulating the efferent motor pattern. To determine the detailed morphology and synaptic connectivity of the intraspinal DA and 5-HT systems in Lampetra fluviatilis and Ichthyomyzon unicuspis, DA and 5-HT antisera were used in light and electron microscopic immunocytochemical experiments. Two main groups of labeled cells were distinguished: DA-containing liquor-contacting (LC) cells distributed along the central canal, and 5-HT+DA-containing multipolar cells located near the midline ventral to the central canal. Both types were synaptically connected with other neuronal elements. The DA-immunoreactive LC cells, which extended a ciliated process into the central canal, received symmetrical synapses from unlabeled terminals containing small synaptic vesicles. The distal process of the LC cells could be traced to the lateral cell column, to the ventral aspect of the dorsal column, or to the ventromedial area. Ultrastructural analysis of DA fibers in these regions showed the presence of labeled terminals containing numerous small synaptic vesicles and a few dense-core vesicles. These terminals formed symmetrical synapses with unlabeled cell bodies and dendrites, with GABA-immunopositive LC cells, and with the multipolar DA+5-HT cells. The multipolar DA+5-HT cells also received input from unlabeled synapses. Intracellular recording from these cells showed that they received excitatory postsynaptic potentials in response to stimulation of fibers in the ventromedial tracts and dorsal roots. The terminals of the multipolar DA+5-HT neurons in the ventromedial spinal cord contained numerous dense-core vesicles and small synaptic vesicles, but no synaptic specializations could be detected. In addition, a small number of larger DA-immunoreactive cells were observed in the lateral cell column at rostral levels. The lamprey spinal cord thus contains distinct populations of synaptically interconnected monoaminergic neurons. Dopamine-containing LC cells synapse onto DA+5-HT-containing multipolar cells, in addition to GABAergic LC cells and unidentified spinal neurons. In contrast, the multipolar cells appear to exert their influence by nonsynaptic mechanisms.

Catecholamine innervation of the piriform cortex: a tracing and immunohistochemical study in the rat

In order to determine the origin of the Catecholamine innervation of the rat piriform cortex (PC), we combined retrograde transport of the B subunit of the cholera toxin (CTb) with tyrosine hydroxylase (TH) immunohistochemistry. A substantial number of CTb retrogradely labeled cells was found in the parabrachial pigmented, paranigral and interfascicular nuclei of the ventral tegmental area and the dorsal part of the locus coeruleus, whereas nearly no labeling was noted in the substantia nigra. Following TH immunohistochemistry on the same sections, most if not all of the CTb labeled cells were also TH immunoreactive. Occasional double-labeled cells were also observed in the anterior part of the raphe dorsal nucleus. As visualized with dopamine β-hydroxylase, dopamine or TH immunohistochemistry, che noradrenaline fibers were homogeneously distributed whereas the dopamine fibers showed rostro-caudal and latero-medial differences. The distribution of TH fibers overlapped both patterns. Our report suggests that the heterogeneous distribution of the DA fibers could support a differential centrifugal modulation of the olfactory information processing throughout the PC.

Cerebellar and striatal dopamine receptors: Effects of reeler and weaver murine mutations

The presence and the binding characteristics of D1 and D2 receptors were investigated in normal-reeler and normal-weaver mutant mice utilizing [3H]spiperone (D2 antagonist), [3H]SKF 38393 (D1 agonist), and [3H]DA as ligands. Analysis of the binding data showed that in the cerebellum there are two binding components for all [3H]ligands. Comparison of the binding constants from cerebellum and striatum showed that in cerebellum the high affinity-low capacity component has similar affinity with that of striatum. The reeler and weaver mutations affected the binding of all ligands: In reeler, total cerebellar specific binding sites for [3H]spiperone and [3H]SKF 38393 decrease significantly (approximately 50% and approximately 70%, respectively), while those for [3H]DA show a small (approximately 10-15%) but not significant decrease. In weaver, total cerebellar specific binding sites for [3H]spiperone, [3H]SKF 38393, and [3H]DA also decrease significantly (approximately 60%, approximately 70%, and approximately 50%, respectively). In reeler striatum [3H]SKF 38393 binding (Bmax) is significantly decreased (approximately 24%), while [3H]spiperone and [3H]DA binding (Bmax) is not affected. In weaver striatum, [3H]SKF 38393 binding is significantly increased (approximately 40%), while [3H]DA binding (Bmax) decreases significantly (approximately 70%). On the basis of the cytoarchitectural aberrations that characterize the cerebellum of these mutants and some well-established information regarding the dopaminergic system of the cerebellum, the above results indicate that in this region a) D1 receptors are mainly localized on granule cells and b) D2 receptors are localized postsynaptically on granule cells and presynaptically on the DA fibers innervating the cerebellum.

Dopamine synthesis precedes dopamine uptake in embryonic rat mesencephalic neurons.

We have measured [3H]dopamine ([3H]DA) uptake and tyrosine hydroxylase-immunopositive immunostaining in cells acutely dissociated from the embryonic ventral mesencephalon (MSC). DA and its metabolites as well as catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) activities were determined in homogenates taken from the MSC and striatum (STR). In the embryonic ventral MSC measurable DA and tyrosine hydroxylase (TH) immunostaining were present as early as embryonic day (E) 12.5. At E14 the number of TH+ neurons was about 50% of the values at E18. In the MSC, DA concentration increased sharply at E16 and reached a plateau before birth that was 10-fold lower than adult values. In the STR, DA was first detected at E16, suggesting that DA fibers reach the STR at this embryonic stage. High-affinity DA uptake appeared in the MSC only at E16, concomitantly with the arrival of DA fibers in the STR, increased sharply between E16 and E18, and reached a plateau before birth. This uptake mechanism was not selective for catecholamine uptake inhibitors. Thus, DA synthesis in the MSC preceded the onset of high-affinity uptake mechanism, which could be correlated to the beginning of striatal DA innervation. Measurable MAO and COMT activities were detected as early as E13 (MSC) and E15 (STR), but not DA metabolites, which appeared later. We conclude that the high-affinity DA uptake mechanism in MSC DA neurons develops coincident with the arrival of DA fibers to the STR. The sharp increase of DA uptake between E16 and E18 is due only in part to an increase in the number of TH+ cells. These results support the hypothesis that in vivo the target STR neurons regulate the maturation of MSC DA cells.

Cografts of adrenal medulla with peripheral nerve enhance the survivability of transplanted adrenal chromaffin cells and recovery of the host nigrostriatal dopaminergic system in MPTP-treated young adult mice

Schwann cells from transected peripheral nerve segments are known to produce nerve growth factor (NGF). We performed adrenal medullary grafts or cografts of adrenal medulla and sciatic nerve into tthe striatum of MPTP-treated young adult mice, and compared the survivability of grafted chromaffin cells and the recovery of intrinsic host DA fibers using computerized image analysis of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers and neurochemical analysis with high performance liquid chromatography (HPLC). Adrenal medullary chromaffin cells cografted with sciatic nerve survived better than those in adrenal grafts alone; host DA fiber recovery was more prominent in mice with cografts than in mice with adrenal grafts alone. A large number of TH-IR surviving cells in cografted mice showed long neuronal processes which were rarely seen in the mice receiving adrenal graft alone. We conclude that cograft of adrenal medulla and sciatic nerve promotes intrinsic host DA fiber recovery better than adrenal medulla grafts alone, and that survivability of grafted chromaffin cell may promote host DA fiber recovery. Adrenal medullary autografts have been used in patients with Parkinson's disease; we suggest that if this approach is to be used in the future, methods to increase the survivability of grafted chromaffin cells, such as co-grafting with pieces of peripheral nerve, be considered to enhance the survivability of the chromaffin cells, which might be closely related to the functional recovery of the patients by this grafting procedure. Of course, such strategies as the present cografting approach must be demonstrated to work in older animals using older donor tissue before proceeding to this next step in humans.

Degeneration and graft-induced restoration of dopamine innervation in the weaver mouse neostriatum: a quantitative radioautographic study of [3H]dopamine uptake

A recently introduced quantitative radioautographic technique was used to characterize the striatal dopaminergic deficit in weaver mutant mice and to evaluate the extent of DA reinnervation resulting from cell suspension grafts of fetal ventral mesencephalic tissue. Brain slices from normal mice and unilaterally grafted weaver mice were incubated in [3H]DA, in the presence of desipramine and pargyline 3-5 months after graft surgery. Semi-thin sections from the fixed and resinembedded slices were subsequently exposed on tritium sensitive film and afterwards dipped in nuclear emulsion for light microscope radioautography. Alternative slices were embedded in Epon for post-embedding tyrosine hydroxylase (TH) immunocytochemistry. The grain density of the film radioautographs matched well the distribution of TH positive fibers. Both methods revealed an almost complete absence of DA axons in the dorsomedial quadrant of the weaver neostriatum and an increasing density of DA innervation towards the ventrolateral areas. In the light microscope radioautographs, only the ventral striatum (i.e. nucleus accumbens and olfactory tubercle) and a narrow ventral and periventricular zone of the caudate-putamen were covered by silver grain clusters typical of DA varicosity labeling. Such labeled varicosities were nevertheless found in reduced numbers the lateral portion of both nucleus accumbens and the olfactory tubercle. The remaining neostriatum was overlaid by diffuse silver grains. suggesting a deficient DA uptake and storage mechanism in the residual DA fibers in this region. Immunocytochemistry using antibodies specific for DA or TH provided further evidence that the residual DA innervation in the weaver neostriatum was biochemically defective. Weaver mice with grafts of ventral mesencephalic tissue in the right neostriatum showed an amphetamine-induced rotational bias to the contralateral side, which was not seen in the sham-operated animals. In contrast to the intrinsic weaver neostriatal DA innervation, DA fibers of graft origin exhibited the normal, clustered type of varicosity labeling. The computerized image analysis of silver grain density in film radioautographs was calibrated by counting these labeled varicosities in selected areas of light microscope radioautographs from the same sections. Results showed a mean DA reinnervation of neostriatal tissue surrounding the graft of about 20%, in some cases up to 80%, of the density seen in wild type mice, with a gradual decrease with distance up to 1-1.4 mm from the graft. The ventral parts of the neostriatum, which contained higher numbers of residual intrinsic DA fibers, were much more sparsely reinnervated than the dorsal and dorsomedial areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Contribution of noradrenergic neurons to the regulation of dopaminergic (D1) receptor denervation supersensitivity in rat prefrontal cortex.

3,4-Dihydroxyphenylethylamine (DA, dopamine) levels in the rat prefrontal cortex were selectively decreased by 52%, leaving noradrenaline (NA) levels unaffected, 4 weeks following restricted bilateral electrolytic lesions of the ventral mesencephalic tegmentum (VMT). These lesions also induced a significant increase in DA-sensitive, but not isoproterenol-sensitive, adenylate cyclase activity in tissue homogenates (+38%). We had shown previously that chemical (6-hydroxydopamine, 6-OHDA) lesions of the VMT destroy both ascending DA and NA fibers but do not alter the D1-receptor density in the prefrontal cortex. In this study, electrolytic lesions of the VMT were combined with bilateral injections of 6-OHDA made laterally in the pedunculus cerebellaris superior to assess the role of NA fibers in the development of D1-receptor supersensitivity. This combined treatment produces a large decrease of cortical NA levels (-95%), an increase in beta-adrenergic-sensitive adenylate cyclase activity (+110%), and a decrease in DA levels (-60%), but does not alter D1-receptor density in the prefrontal cortex. These results indicate that the development of D1-receptor supersensitivity in the prefrontal cortex following electrolytic lesion of the VMT depends on the presence of an intact NA innervation.

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.

Physiopathology of experimental Parkinsonism in the monkey.

Postural or Parkinson-like tremor, which results from the impairment of mechanisms which are predominantly lateralized in the brain, is most likely related to the combined impairment of the dopaminergic nigrostriatal pathway and the corresponding rubro-olivo-cerebello-rubral loop (without excluding the possiblity that other nervous mechanisms interconnected with these structures may represent an alternative disturbance). The integrity of the internal division of the pallidum and the ventrolateral area of the thalamus and their efferent fibers as well as the motor cortex and certain of its cortico-subcortico-spinal pathways (Figures 1 and 2) is apparently an essential feature for the elaboration of the rhythmic bursts associated with the appearance of postural tremor. The integrity of the spinal sensory roots and the rubro-tegmentospinal tract is not a prerequisite for the expression of postural tremor, a condition which seems essential for the production of rigidity. The latter facts suggest that the disturbances which subserve these two types of motor impairment, often concomitantly present in Parkinsonism, partially involve the impairment of different mechanisms although the loss of the DA fibers originating in the substantia nigra and ending in the neostriatum (Figure 1) appears to represent a disturbance common to both types of disorders. Bradykinesia which may be associated with an impairment of catecholamine metabolism (and more especially the neostriatal DA mechanisms) on both sides of the brain may also result from bilateral lesions of the pallidum or of its outflow corresponding, in the main, to the pallidothalamic fibers ending in the ventrolateral thalamus. The latter types of lesion most likely exclude the influence of the monoaminergic, cholinergic and gabaminergic activities normally originating in the striopallidal system and influencing the activity transmitted to other CNS mechanisms. Severe akinesia, however, apparently depends on more profound and generalized disturbances of brain monoamine metabolism with or without the involvement of other ill-defined mechanisms. At any rate the impairment of the brain DA mechanisms (and especially those of the neostriatum) seems to represent a major feature in the production of the Parkinsonian type of akinesia. Further work is needed to establish the relative importance of the loss of catecholaminergic mechanisms other than those of the neostriatum in the production of akinesia.

Depletion of catecholamines in vivo induced by electrical stimulation of central monoamine pathways

Intermittent electrical stimulation of central monoamine pathways has been carried out in anesthetized rats pretreated with inhibitors of amine synthesis. Depletion of catecholamines, as assessed by the histochemical fluorescence technique, was observed in DA and NA nerve terminal systems in various areas of the brain according to the area of stimulation. Without inhibition of amine synthesis no amine depletion was observed. Stimulation of the dorsal bundle (a collection of NA-containing fibere probably mainly arising from the locus coeruleus (group A6)) resulted in amine depletion mainly of NA terminals in the cortex cerebri and the hippocampal formation. Stimulation of the ventral NA bundle in the mesencephalon resulted in amine depletion mainly of NA terminals in the ventral diencephalon ( e.g. hypothalamus) and the ventral parts of the limbic system. Similarly, stimulation of the DA axons arising from the zona compacta of the substantia nigra (group A9) led to depletion of amine from DA nerve terminals in the neostriatum. Stimulation in the A10 cell body area resulted in depletion of amines from NA (hypothalamus + ventral parts of the limbic system) and/or DA (limbic forebrain and/or neostriatum) nerve terminals in the tel- and diencephalon. It is suggested that these effects are the results of stimulation of DA cell bodies and fibers in this area and NA fibers of passage originating from the ventral NA bundle. As the stimulus parameters used in this study were determined in separate experiments on intracerebral self-stimulation, it seems likely that, with certain electrode placements, the levels of stimulation used in such behavioral experiments may be effective in releasing central catecholamines.