Tamoxifen Diminishes Methamphetamine-Induced Striatal Dopamine Depletion in Intact Female and Male Mice

It has been demonstrated that the nigrostriatal dopaminergic system of male mice is more sensitive to the neurotoxic effects of methamphetamine (MA). The basis for this difference can be related to oestrogen, which has the capacity to function as a neuroprotectant against neurotoxins that target the nigrostriatal dopaminergic system. We examined the effects of the anti-oestrogen, tamoxifen (TMX), upon MA-induced neurotoxicity of the nigrostriatal dopaminergic system in intact female and male CD-1 mice. Striatal dopamine concentrations of TMX-treated female and male mice receiving MA were significantly greater than mice receiving MA alone. In female, but not male, mice, oestrogen treatment also resulted in greater striatal dopamine concentrations compared to mice receiving MA alone. Interestingly, male mice treated with oestrogen were particularly sensitive to the acute toxic effects of MA and displayed no evidence of nigrostriatal neuroprotection. The dihydroxyphenylacetic acid/dopamine ratios following MA for female and male mice treated with TMX or females treated with oestrogen were significantly reduced compared to MA-treated mice and oestrogen + MA-treated male mice. No differences among the treatment groups were obtained for dopamine in the hypothalamus or olfactory bulb. These data demonstrate that TMX treatment of intact female and male mice diminishes striatal dopamine depletions to the nigrostriatal dopaminergic neurotoxin, MA. Oestrogen also displayed this capacity when administered to female, but accentuated acute toxicity in male mice. These effects are relatively specific for the nigrostriatal dopaminergic system. Such data suggest that TMX can function as a nigrostriatal dopaminergic neuroprotectant against MA-induced neurotoxicity in intact female and male mice.     

Dopamine transporter loss and clinical changes in MPTP-lesioned primates

Single photon emission computed tomography (SPECT) and the dopamine (DA) transporter tracer, 2 beta-carboxymethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT), were used to determine DA transporter density in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-lesioned monkeys with varying degrees of parkinsonism. The clinical stage of parkinsonism corresponded to SPECT measures of striatal DA transporter density suggesting that more severe parkinsonism was associated with a greater degree of dopaminergic terminal degeneration. These findings are similar to those reported earlier using positron emission tomography (PET) and the DA metabolism tracer, 6-[18F]fluoro-L-m-tyrosine (FMT), indicating that both are good methods for evaluating nigrostriatal degeneration in MPTP primate models.     

Regulation of connexin-43, GFAP,and FGF-2 is not accompanied by changes in astroglial coupling in MPTP-lesioned,FGF-2-treated Parkisonian mice

Basic fibroblast growth factor (bFGF; FGF-2) has potent trophic effects on developing and toxically impaired midbrain dopaminergic (DAergic) neurons which are crucially affected in Parkinson's disease. The trophic effects of FGF-2 are largely indirect, both in vitro and in vivo, and possibly involve intermediate actions of astrocytes and other glial cells. To further investigate the cellular and molecular mechanisms underlying the restorative actions of FGF-2, and to analyse in more detail the changes within astroglial cells in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned striatum, we have studied striatal expression and regulation of connexin-43 (cx43), the principal gap junction protein of astroglial cells, along with the expression of glial fibrillary acidic protein (GFAP), FGF-2, and functional coupling. Our results show an immediate, yet transient increase in cx43 mRNA, and a sustained increase in FGF-2 mRNA, GFAP-positive cells, and cx43-immunoreactive punctata following the MPTP lesion, without any induction of functional coupling between astrocytes and other glial cells as revealed by dye coupling of patched cells. Unilateral administration of FGF-2 in a piece of gelfoam caused a further increase in cx43-positive punctata immediately adjacent to the implant, which was more pronounced than after application of a gelfoam containing the non-trophic control protein in cytochrome C. These changes were parallelled by a small increase in cx43 protein determined by Western blot, but not by alterations in the coupling state of cells in the vicinity of the gelfoam implant. Although our data indicate that MPTP and exogenous FGF-2 may alter expression and protein levels of cx43, they do not support the notion that increases in cellular coupling may underly the trophic and widespread actions of FGF-2 in the MPTP-model of Parkinson's disease. © 1996 Wiley-Liss, Inc.     

Striatal AMPA receptor binding is unaltered in the MPTP-lesioned macaque model of Parkinson's disease and dyskinesia

Long-term levodopa or dopamine agonist treatment in the MPTP-lesioned primate model of Parkinson's disease elicits dyskinesia, which is phenotypically similar to levodopa-induced dyskinesia in patients with Parkinson's disease. AMPA receptor antagonists have previously been shown to have both anti-parkinsonian and anti-dyskinetic actions in MPTP-lesioned primates, suggesting that AMPA receptor transmission is functionally overactive under these conditions. In this study, we investigated the level of striatal AMPA receptor binding in the MPTP lesioned primate using the selective AMPA ligand (3)H-(S)-5-fluorowillardiine. AMPA receptor binding was studied in non-parkinsonian, non-dyskinetic parkinsonian, and dyskinetic macaques. Striatal AMPA receptor binding was not different in any of the treatment groups (P > 0.05). Although AMPA receptor-mediated transmission is functionally overactive in Parkinson's disease and dyskinesia, changes in striatal AMPA receptor levels are not likely to be the cause of such movement disorders.     

Effects of FGF-1 and FGF-2 on GD3 immunoreactive spinal neuroepithelial cells

Embryonic central nervous system neuroepithelial cells are a transient population of cells that give rise to neuronal and glial progenitors. In the E12–E16 embryonic rat spinal neural tube we have identified neuroepithelial cells as radially oriented cells expressing the GD3 ganglioside as recognized by the monoclonal anti-GD3 ganglioside antibodies, R24 and LB1. In vitro, neuroepithelial cells, which migrate from the ventral aspect of E12 rat lumbosacral neural tube explants, also express GD3 ganglioside immunoreactivity, thus permitting their distinction from neural crest cells (NCC) which migrate from the dorsal aspect of such explants. Fibroblast growth factor-1 (FGF-1, acidic FGF) and FGF-2 (basic FGF) increase the migration of neuroepithelial cells and the extent to which they incorporate the thymidine analogue bromodeoxyuridine (BrdU). They do not, however, alter the rate at which these migrating neuroepithelial cells undergo cell death. Previous observations established the actions of FGF-1 and FGF-2 on neuronal and glial cells. The present study indicates that these growth factors also influence the motility and proliferation of progenitor cells at a developmental stage which precedes their divergence into neuronal and glial lineages. © 1996 Wiley-Liss, Inc.     

Regulation of astrocyte GFAP expression by TGF-β1 and FGF-2

Astrocytes play a critical role in the development of the CNS and its response to injury and disease. A key indicator of astrocyte activation is the increased accumulation of intermediate filaments composed of glial fibrillary acidic protein (GFAP). Treatment of astrocytes in vitro with transforming growth factor-β1 (TGF-β1) produced little morphological change, but resulted in a significant increase in GFAP mRNA and protein. Treatment with basic fibroblast growth factor (FGF-2) produced a dramatic change from a polygonal to a stellate morphology, and resulted in a significant decrease in GFAP mRNA and protein. FGF-2 also inhibited the TGF-β1-mediated increase in GFAP mRNA and protein. Cycloheximide did not block the effects of TGF-β1 or FGF-2 on GFAP mRNA levels, but blocked the inhibitory effects of FGF-2 on the TGF-β1-mediated increase in GFAP expression. All effects of FGF-2 were blocked by co-incubation with 5′-methylthioadenosine, a specific inhibitor of FGF-2-induced tyrosine kinase activity and FGF receptor (FGFR) autophosphorylation. We also examined astrocyte expression of FGFR, and demonstrate the presence of FGFR 1 and 2, and lower levels of FGFR 3. Our results demonstrate that TGF-β1 and FGF-2 cause differential effects on the astrocyte cytoskeleton and morphology, suggesting an uncoupling of process outgrowth from GFAP synthesis. GLIA 22:202–210, 1998. © 1998 Wiley-Liss, Inc.     

Interleukin 6 in Intact and Injured Mouse Peripheral Nerves

The multifunctional cytokine interleukin 6 (IL-6) has direct growth, survival and differentiation effects on peripheral and central neurons. Furthermore, it can modulate the production by non-neuronal cells of other cytokines and growth factors, and thereby affect nerve cells indirectly. We have studied IL-6 expression and production in intact and injured peripheral nerves of C57/BL/6NHSD mice, which display the normal rapid progression of Wallerian degeneration. The IL-6 mRNA was detected in nerves degenerating in vitro or in vivo , but not in intact nerves. In vitro - and in vivo -degenerating nerve segments and neuroma nerve segments synthesized and secreted IL-6. The onset of IL-6 production was rapid and prolonged. It was detected as early as 2 h after injury and persisted for the entire period of 21 days tested after the injury. Of the non-neuronal cells that reside in intact and injured nerves, macrophages and fibroblasts were the major contributors to IL-6 production. We also studied IL-6 production in intact and injured nerves of mutant C57BL/6-WLD/OLA/NHSD mice, which display very slow progression of Wallerian degeneration. Injured nerves of C57BL/6-WLD/OLA/NHSD mice produced significantly lower amounts of IL-6 than did rapidly degenerating nerves of C57/BL/6NHSD mice.     

The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain

Neurogenesis occurs in two regions of the adult brain, namely, the subventricular zone (SVZ) throughout the wall of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampal formation. Adult neurogenesis requires several neurotrophic factors to sustain and regulate the proliferation and differentiation of the adult stem cell population. In the present review, we examine the cellular and functional aspects of a trophic system mediated by fibroblast growth factor-2 (FGF-2) and its receptors (FGFRs) related to neurogenesis in the SVZ and SGZ of the adult rat brain. In the SVZ, FGF-2 is expressed in GFAP-positive cells of SVZ but is not present in proliferating precursor cells, which instead express FGFR-1 and FGFR-2, but not FGFR-3 mRNA, although expressed in the SVZ, and FGFR-4. Therefore, it seems that in the SVZ FGF-2 may be released by GFAP-positive cells, different from the precursor cell lineage, and via volume transmission it reaches the proliferating precursor cells. FGFR-1 mRNA is also expressed in the SGZ and is localized in BrdU-labeled precursor cells, whereas FGFR-2 and FGFR-3 mRNA, although expressed in the SGZ, are not located within proliferating precursor cells. An aged-related decline of proliferating precursor cells in the SVZ and DG of old rats has been well documented, and there is the suggestion that in part it could be the consequence of alterations in growth factor expression levels. Thus, the old precursors may respond to growth factors, suggesting that during aging the basic components for neuronal precursor cell proliferation are retained and the capacity to increase neurogenesis after appropriate stimulation is still preserved. In conclusion, the trophic system mediated by FGF-2 and its receptors contributes to create an important micro-environmental niche that promotes neurogenesis in the adult and aged brain. This article is dedicated to the special issue Brain Plasticity: Aging and Neuropychiatric Disorders.     

Cholecystokinin Octapeptide In Vitro and Ex Vivo Strongly Modulates Striatal Dopamine D2 Receptors in Rat Forebrain Sections

Receptor autoradiographic experiments together with the filter wipe-off technique were performed to investigate the effects of cholecystokinin octapeptide (CCK-8) on dopamine D₂ receptors. In vitro studies showed that 1 nM CCK-8 significantly increased the K_D value of binding sites for the D₂ agonist [³H]N-propylnorapomorphine (NPA) in the rostral and caudal parts of the nucleus accumbens by 48 and 148% respectively. In contrast, 1 nM CCK-8 significantly decreased the IC₅₀ value of dopamine for binding sites for the D₂ antagonist [¹²⁵I]iodosulpride in the rostral and caudal parts of the caudate-putamen by 46 and 56% respectively, and in the rostral and caudal parts of the nucleus accumbens (areas of CCK-dopamine coexistence) by 57 and 75% respectively. Ex vivo studies demonstrated that 30 min after an intraventricular injection of 1 nmol/rat CCK-8 the K_D value of [³H]NPA binding sites in the caudal part of the forebrain and the IC50 value of dopamine for [¹²⁵I]iodosulpride binding sites in the caudal part of the nucleus accumbens were significantly increased by 160% and decreased by 77% respectively. These results indicate for the first time that in sections CCK-8 in vitro and ex vivo can strongly regulate D₂ receptor affinity in the striatum. The present studies also provide evidence for stronger modulation of D₂ receptors by CCK-8 in the area of CCW-dopamine coexistence in the nucleus accumbens than in other basal ganglion areas, supporting the existence of CCWD₂ receptor interactions in cotransmission. The stronger interactions found in sections than in membrane preparations may indicate the requirement of intracellular mechanisms and/or a more intact membrane structure for optimal receptor-receptor interactions.     

Dopamine differentiation factors increase striatal dopaminergic function in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned mice

We have previously shown that muscle-derived differentiation factors (MDF) and human recombinant acidic fibroblast growth factor (aFGF) have beneficial behavioral and neurochemical effects on the nigrostriatal dopaminergic neurons of 6-hydroxy-dopamine (6-OHDA)-lesioned rats (Jin and Iacovitti: Neurobiol Dis 2:1–12, 1995). In the present study, we determined the effects of similar treatments on mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Five days after unilateral striatal infusion of MDF or aFGF into MPTP-lesioned mice, striatal tyrosine hydroxylase (TH) activity and dihydroxyphenylacetic acid (DOPAC) levels were bilaterally increased (20–35%) compared to untreated (lesion only) or control (phosphate buffered saline + bovine serum albumin) mice. These increases, however, were not accompanied by change in dopamine (DA) levels, indicating an elevation of DA synthesis (TH/DA) and turnover (DOPAC/DA). The present findings that MDF and aFGF may have neurochemical effects in vivo on the lesioned nigrostriatal dopaminergic system suggest their potential pharmacological role in the treatment of Parkinson's disease. © 1996 Wiley-Liss, Inc.     

The High Sensitivity to Rotenone of Striatal Dopamine Uptake Suggests the Existence of a Constitutive Metabolic Deficiency in Dopaminergic Neurons From the Substantia Nigra

The toxicity of the 1-methyl-4-phenylpyridinium ion (MPP⁺), an inhibitor of complex I of the respiratory chain, on nigrostriatal dopaminergic neurons contrasts with its relative inefficiency towards other catecholaminergic cell populations in spite of their ability to accumulate this neurotoxin through their high-affinity uptake system. A constitutive metabolic deficiency of the nigrostriatal dopaminergic neurons could account for their particular vulnerability to MPP⁺. In order to substantiate this hypothesis, we compared the inhibitory effects of rotenone, an inhibitor of mitochondrial oxidative phosphorylation, on the uptake of dopamine, serotonin, noradrenaline and GABA in mouse striatal synaptosomes, and of dopamine, serotonin and GABA in cultured mesencephalic neurons. In both preparations, the uptake of dopamine was much more affected than that of other neurotransmitters by rotenone. This result was confirmed using two other unrelated inhibitors of oxidative phosphorylation. Moreover, dopamine uptake in synaptosomes from the dorsolateral striatum was more sensitive to rotenone than uptake in synaptosomes from the nucleus accumbens. This indicates that intrinsic metabolic properties of the nigrostriatal dopaminergic neurons may explain the strong inhibition by rotenone of striatal dopamine uptake. Altogether, these results suggest that a constitutive metabolic deficiency could account, at least in part, for the selective vulnerability of the nigrostriatal dopaminergic pathway to the action of the neurotoxin MPP⁺.     

Administration of FGF-2 to embryonic mouse brain induces hydrocephalic brain morphology and aberrant differentiation of neurons in the postnatal cerebral cortex

Fibroblast growth factor-2 (FGF-2) was injected into mouse cerebral ventricles at embryonic day (E) 14 in utero and its effects on developing brain morphology and expression of various cell- or differentiation-associated protein markers in the cerebral cortex were examined. High doses of FGF-2 (200 or 300 ng) caused encephalic alternations such as deformation of the calvarium, enlargement of the ventricular spaces, and thinning of the cerebral cortex. There was no gross abnormality in the alignment of the cerebral neuronal layers, however, both cell number and cell density of the upper layers (II/III) and the lower layers (IV-VI) of the cerebral cortex were increased. Brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase, nestin, and microtubule-associated protein 2 were aberrantly or ectopically expressed in the deep areas of the cerebral cortex. A substantial number of these cells coexpressed these antigens. These observations demonstrate that a subpopulation of neurons in the cortical deep layer abnormally differentiated or partly sustained their immature state following a single administration of FGF-2 at E14. Developmental analysis of localization of BDNF-positive cells suggested that the abnormality started around P5. Furthermore, cell migration was not affected by FGF-2 administration. FGF-2 seems to play predominant roles in the proliferation of neuronal precursors and in neuronal differentiation in the developing mouse cerebral cortex even at relatively late stages of brain neurogenesis.     

Dopamine: Mesocortical vs nigrostriatal uptake in isolated fighting mice and controls

Acute isolation-induced fighting in mice produced a striking ‘dose-dependent’ increase in K_m and V_max for DA uptake in mesocortical nerve endings (synaptosomes) but no significant changes for these uptake constants in nigrostriatal terminals. The selective response of the mesocortical system to fighting is similar to that reported for other affective paradigms and stress.Several controls were carried out simultaneously to sort out neurotransmitter responses due to aggressive behavior from those due to secondary behaviors which accompany fighting. Non-significant or borderline increases in K_m and V_max for mesocortical DA uptake were noted in (i) animals permitted to witness fighting but prevented from participating, (ii) those subjected to the stress of a mock-fighting situation and (iii) animals simply handled by the investigator, but in no case were the control values increased to the magnitude produced by fighting. (iv) Group-housed animals showed no mesocortical response when placed in the arena with isolated mice. They retired to the corners and would not fight. These mild to negative control findings indicate that the marked cortical responses seen in the fighting animals are not due to (i) anticipation of the fight, (ii) physical stress and motor activity, (iii) placing the animals in the combat arena, or (iv) isolation. Significant changes in nigrostriatal DA reactivity were noted only in the isolated animals prevented from fighting where there was a decrease in K_m and V_max for DA uptake.     

The NR2B-selective NMDA receptor antagonist CP-101,606 exacerbates L-DOPA-induced dyskinesia and provides mild potentiation of anti-parkinsonian effects of L-DOPA in the MPTP-lesioned marmoset model of Parkinson's disease

In Parkinson's disease (PD), degeneration of the dopaminergic nigrostriatal pathway leads to enhanced transmission at NMDA receptors containing NR2B subunits. Previous studies have shown that some, but not all, NR2B-containing NMDA receptor antagonists alleviate parkinsonian symptoms in animal models of PD. Furthermore, enhanced NMDA receptor-mediated transmission underlies the generation of L-DOPA-induced dyskinesia (LID). The subunit content of NMDA receptors responsible for LID is not clear. Here, we assess the actions of the NMDA antagonist CP-101,606 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmoset model of Parkinson's disease. CP-101,606 is selective for NMDA receptors containing NR2B subunits, with higher affinity for NR1/NR2B complexes compared to ternary NR1/NR2A/NR2B complexes. CP-101,606 had no significant effect on parkinsonian symptoms when administered as monotherapy over a range of doses (0.1-10 mg/kg). CP-101,606 provided a modest potentiation of the anti-parkinsonian actions of L-DOPA (8 mg/kg), although, at doses of 1 and 3 mg/kg, CP-101,606 exacerbated LID. Results of this study provide further evidence of differences in the anti-parkinsonian activity and effects on LID of the NR2B subunit selective NMDA receptor antagonists. These distinctions may reflect disparities in action on NR1/NR2B as opposed to NR1/NR2A/NR2B receptors.     

FGF-2 deficiency does not alter vulnerability of the dopaminergic nigrostriatal system towards MPTP intoxication in mice

Fibroblast growth factor 2 (FGF-2) was the first growth factor discovered that exerted prominent protective and regenerative effects in an animal model of Parkinson's disease, the MPTP-lesioned dopaminergic nigrostriatal system. To address the putative physiological relevance of endogenous FGF-2 for midbrain dopaminergic neurons, we have analysed densities of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) and TH-positive fibers in the striatum and amygdala of adult FGF-2-deficient mice. We found that densities of TH-immunoreactive (ir) cells in the SN as well as densities of TH-ir fibers in the striatum and amygdala were unaltered as compared with wild-type littermates. There is evidence to suggest that growth factor deficits do not become apparent unless a system is challenged in a lesioning paradigm. We therefore tested the ability of the nigrostriatal system with respect to its ability to cope with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) intoxication. Treatment with 20 mg/kg MPTP on three consecutive days reduced dopamine levels in the striatum by about 80%. Densities of TH-positive neurons in the SN were reduced by 71%. However, both parameters did not significantly differ between FGF-2(-/-) mice and wild-type littermates. Our results therefore suggest that FGF-2, despite its prominent pharmacological potency as a neurotrophic factor for the dopaminergic nigrostriatal system, is not crucial for maintaining its structural integrity and ability to cope with MPTP intoxication.     

EGF和FGF-2促神经干细胞增殖与分化研究

目的 建立人胚胎大脑神经干细胞的分离培养方法并观察在不同诱导因子作用下细胞分化的生物学特性。方法 将经培养的人胚胎大脑神经干细胞分为4组,在不同培养液中培养、分化:(1)DMEM/F-12标准培养液组;(2)标准培养液+表皮生长因子(EGF)组;(3)标准培养液+碱性成纤维细胞生长因子-2(FGF-2)组;(4)标准培养液+表皮生长因子+碱性成纤维细胞生长因子-组。用定量RT-PCR方法及免疫组织化学染色方法鉴定神经干细胞的原始特性、端粒酶活性以及不同诱导因子对神经干细胞分化特性的影响。结果 于培养第6d,标准培养液中的脑细胞死亡;而在加入EGF和FGF-2的培养液中则形成神经干细胞球体,而且增殖细胞核抗原染色和神经干细胞巢蛋白表达均呈阳性反应;其端粒酶活性为63％,低于瘤细胞而高于正常脑组织。神经干细胞分化后经胶质纤维酸性蛋白、神经微丝、突触素、微管相关蛋白-2及神经细胞核抗原染色显示,分化的神经元最高值达(18±2.5)％。不同诱导因子对不同传代的细胞球体内神经干细胞的促增殖作用差异有显著性意义(均P<0.05)。结论 EGF和EGF-2均具有促进神经干细胞存活、增殖和分化的作用。