Effect of acute and chronic deprivation of catecholaminergic system activity induced by 6-hydroxydopamine on the behavior of the rat

On 202 male rats of Wistar line, a study was carried out of the effect of chronic and acute deprivations of the brain CA-systems activity resulting from administration of 6-OHDA on investigating behaviour and learning. Chronic deprivation of CA-systems activity by neonatal administration of 6-OHDA (100 mg/kg subcutaneously) and their acute deprivation by intracerebral administration of 6-OHDA to adult rats (150 mkg in each lateral ventriculus) was accompanied by similar deep changes of behaviour. Both forms of deprivation reduced the investigating activity of the animals in the open field. In both cases, the above 6-OHDA dozes sharply impeded the learning of animals with emotionally negative reinforcement, with no significant influence on learning with emotionally positive reinforcement. Both forms of deprivation of CA-systems activity weakened the reaction of frustration elicited by a sharp reduction of food reinforcement.     

Effects of 6-hydroxydopamine on primary cultures of substantia nigra: specific damage to dopamine neurons and the impact of glial cell line-derived neurotrophic factor

6-Hydroxydopamine (6-OHDA)-induced loss of dopamine (DA) neurons has served to produce an animal model of DA neuron loss in Parkinson's disease. We report here the use of 6-OHDA to produce an in vitro model of this phenomena using dissociated cultures prepared from neonatal rat mesencephalon. Cultures were exposed to 6-OHDA (40-100 microm, 15 min) in an antioxidant medium, and DA and GABA neurons evaluated by immunocytochemistry. 6-OHDA induced morphological and biochemical signs of cell death in DA neurons within 3 h, followed by loss of tyrosine hydroxylase immunoreactive neurons within 2 days. In substantia nigra (SN) cultures, DA neurons were much more affected by 6-OHDA than were GABA neurons. In contrast, DA neurons from the ventral tegmental area were only lost at higher, non-specific concentrations of 6-OHDA. The effects of 6-OHDA on nigral DA neurons were blocked by inhibitors of high affinity DA transport and by z-DEVD-fmk (150 microm), a caspase inhibitor. Glial cell line-derived neurotrophic factor (GDNF) treatment reduced TUNEL labeling 3 h after 6-OHDA exposure, but did not prevent loss of DA neurons at 48 h. Thus, 6-OHDA can selectively destroy DA neurons in post-natal cultures of SN, acting at least in part by initiating caspase-dependent apoptosis, and this effect can be attenuated early but not late by GDNF.     

Effect of 6-hydroxydopamine on brain and blood catecholamine, ammonia, and amino acid metabolism in rats subjected to high pressure oxygen induced convulsions

Effects of 6-hydroxydopamine (6-OHDA) on rat brain and blood adrenaline (A), noradrenaline (NA), ammonia (NH3), gamma-aminobutyric acid (GABA), and amino acid metabolism prior to and after high pressure oxygen (OHP) induced convulsions have been studied. 6-OHDA reduces GABA and glutamate (Glu) rior to OHP exposure in rat brain so that the concentration is even equal to that seen in nondrugged animals after convulsion. Concomitantly, 6-OHDA reduces the latency of OHP-induced convulsion significantly, and increases brain NH3, glutamine, and asparagine significantly. Although 6-OHDA, in increasing dosage, elevates blood A concentration, convulsion produces a significant further increase in A. Blood NA was not significantly changed in drugged, convulsed animals and was much less than blood NA concentrations in nondrugged convulsed animals. Increasing doses of 6-OHDA also increase NH3 in the blood significantly and convulsion increases its concentration further. Latency of convulsion seems to be related to certain monoamine levels since in some drugged animals where A and total catecholamines are still reduced 96 h after the first of two doses of 6-OHDA, NA concentrations are recovered to relatively normal and the convulsion latency time is also increased although it remains significantly abbreviated from undrugged animals' convulsion time. Low brain GABA levels seem to be a prime effector of convulsive activity.     

6-OHDA制备的帕金森病大鼠黑质神经元的超微结构改变

目的观察6-羟多巴胺(6-OHDA)单侧注射制备的帕金森病(PD)大鼠多巴胺(DA)能神经元的超微结构改变。方法单侧微量注射6-OHDA制备PD大鼠模型,用免疫荧光组织化学方法观察正常侧与6-OHDA注射侧黑质酪氨酸羟化酶(TH)阳性神经细胞及神经纤维的变化;并利用免疫电镜技术观察大鼠正常侧与注射侧黑质致密部DA能神经元的超微结构。结果免疫荧光法显示注射侧黑质致密部TH阳性细胞数和网状部TH阳性纤维面积与正常侧的百分比平均值分别为21.83%,23.19%。免疫电镜显示:TH免疫反应阳性产物表达于PD大鼠正常侧DA能神经元的高尔基复合体质膜面及胞质内,电子密度较高,注射侧很少见或几乎未见,且注射侧线粒体嵴有不同程度的溶解,呈空泡样变或髓样变,粗面内质网脱颗粒。结论6-OHDA可引起DA能神经元发生凋亡的超微结构改变。     

Characteristics of rat training on emotionally varied reinforcement under conditions of an oriented change in the level of brain serotonin and noradrenaline]

The effect of serotonin and noadrenaline precursors on the training of the animals on emotionally different reinforcement was studied. 5-OTP (10 mg/kg) facilitated the training of rats on the food reinforcement, but aggravated it on the pain reinforcement. D, L-DOPA (20 mg/kg) facilitated the training on the food reinforcement. The influence of precursors on the peculiarity of the serotonin and noradrenaline distribution in the brain structures of trained animals was also determined by the biological significance of the reinforcement used in the training.     

Desmethylimipramine pretreatment prevents 6-hydroxydopamine induced somatostatin receptor reduction in the rat hippocampus.

Several studies have shown anatomical and functional interconnections between catecholaminergic and somatostatinergic systems. To assess whether somatostatin (SS) may act presynaptically on catecholamine neurons, SS receptors were measured using radioligand test-tube binding assays on synaptosomes from hippocampus and frontoparietal cortex--areas that are innervated by catecholaminergic neurons with different densities and that have a high number of SS receptors--from control and 6-hydroxydopamine (6-OHDA)-treated rats. Intracerebroventricular (i.c.v.) injection of the catecholamine neurotoxin 6-OHDA (0.78 mg free base/kg of body weight in saline with 0.1% ascorbic acid) lowered hippocampal and frontoparietal cortical noradrenaline (NA) and dopamine (DA) levels at 1 week following the injection. Pretreatment of rats with desmethylimipramine (DMI) (40 mg/kg, intraperitoneal) prevented the drop in NA levels, but was not effective in attenuating DA depletion in the two brain areas studied. Treatment with 6-OHDA lowered the number of 125I-Tyr11-SS receptors in the hippocampus (130 +/- 19 vs. 266 +/- 16 fmol/mg protein, P < 0.001), whereas in the frontoparietal cortex a non significant 20% reduction in receptor number was found. The dissociation constants of 125I-Tyr11-SS binding to synaptosomes from frontoparietal cortex (0.65 +/- 0.06 vs. 0.60 +/- 0.04, P not significant) and hippocampus (0.44 +/- 0.04 vs. 0.63 +/- 0.14, P not significant) were similar in control and treated groups. Pretreatment with DMI reversed up to 80% of the effect of 6-OHDA on hippocampus SS receptors. DMI alone had no observable effect on the number and affinity of SS receptors. The 6-OHDA and the DMI treatment did not affect SLI levels in the brain areas studied. These results suggest that a portion of the hippocampal SS receptors may be localized presynaptically on the noradrenergic and dopaminergic nerve terminals.     

Human albumin prevents 6-hydroxydopamine-induced loss of tyrosine hydroxylase in in vitro and in vivo

Human albumin has recently been demonstrated to protect brain neurons from injury in rat ischemic brain. However, there is no information available about whether human albumin can prevent loss of tyrosine hydroxylase (TH) expression of dopaminergic (DA) neurons induced by 6-hydroxydopamine (6-OHDA) toxicity that is most commonly used to create a rat model of Parkinson's disease (PD). In the present study, two microliters of 1.25% human albumin were stereotaxically injected into the right striatum of rats one day before or 7 days after the 6-OHDA lesion in the same side. D-Amphetamine-induced rotational asymmetry was measured 7 days, 3 and 10 weeks after 6-OHDA lesion. We observed that intrastriatal administration of human albumin significantly reduced the degree of rotational asymmetry. The number of TH-immunoreactive neurons present in the substantia nigra was greater in 6-OHDA lesioned rats following human albumin-treatment than non-human albumin treatment. TH-immunoreactivity in the 6-OHDA-lesioned striatum was also significantly increased in the human albumin-treated rats. To examine the mechanisms underlying the effects of human albumin, we challenged PC12 cells with 6-OHDA as an in vitro model of PD. Incubation with human albumin prevented 6-OHDA-induced reduction of cell viability in PC12 cell cultures, as measured by MTT assay. Furthermore, human albumin reduced 6-OHDA-induced formation of reactive oxygen species (ROS) and apoptosis in cultured PC12 cells, as assessed by flow cytometry. Western blot analysis showed that human albumin inhibited 6-OHDA-induced activation of JNK, c-Jun, ERK, and p38 mitogen-activated protein kinases (MAPK) signaling in PC12 cultures challenged with 6-OHDA. Human albumin may protect against 6-OHDA toxicity by influencing MAPK pathway followed by anti-ROS formation and anti-apoptosis.     

Nicotine's attenuation of body weight involves the perifornical hypothalamus

Previously we showed that intermittent administration of nicotine (NIC) in the dark phase decreased food intake and body weight and this could be blocked when the NIC receptor antagonist mecamylamine was infused into the fourth ventricle. Catecholaminergic neurons adjacent to the fourth ventricle contain NIC receptors and directly innervate the perifornical hypothalamus (PFH) which has been shown to be involved in regulation of feeding. This study explored whether NIC regulates feeding behavior by modulating catecholaminergic input to the PFH. Epinephrine and norepinephrine neuronal input was ablated within the PFH by infusion of 6-hydroxydopamine hydrobromide (6-OHDA), while bupropion was infused to protect dopaminergic neurons. After recovery of body weights to pre-surgery levels, food intake, meal size, meal number and body weight were measured after intermittent NIC injections. The results showed the PFH lesioned animals did not exhibit the typical prolonged drop in food intake, meal size and body weight normally associated with NIC administration. High performance liquid chromatography analyses demonstrated that compared to control rats, 6-OHDA administration significantly reduced PFH norepinephrine and epinephrine levels, but not dopamine levels. These results are consistent with NIC reducing food intake in part by acting through catecholaminergic neurons within or extending through the PFH.     

Immortalized dopamine neurons: A model to study neurotoxicity and neuroprotection

6-Hydroxydopamine (6-OHDA) causes selective degeneration of dopaminergic neurons in the rat brain and has been used to produce an animal model of Parkinsonism. Recently, a clonal line of immortalized dopamine (DA) neurons (1RB3AN27), which expresses varying levels of tyrosine hydroxylase, dopamine transporter, neuron specific enolase, and nestin, was established. These DA neurons reduce behavioral deficits in 6-OHDA-lesioned rats. The relative sensitivity of fetal and adult neurons to potential neurotoxins is not well defined. The availability of immortalized DA neurons provides a unique opportunity to compare the relative neurotoxicity of 6-OHDA in differentiated and undifferentiated DA neurons in vitro and identify neuroprotective agents. Our results showed that 6-OHDA treatment for 24 hr decreased the viability of undifferentiated and differentiated immortalized DA neurons in vitro, as determined by the MTT assay, and increased the rate of apoptosis in differentiated DA neurons. The differentiated DA neurons (IC50 = 33 microM) were about 2-fold more sensitive to 6-OHDA than undifferentiated DA neurons (IC50 = 75 microM) in cell culture. Similarly, the differentiated DA neurons were more sensitive to another neurotoxin, 1-methyl-4-phenylpyridinium (MPP+), which is commonly used to induce Parkinsonism in animal models, than were the undifferentiated DA neurons in culture. Among growth factors tested, only glial cell line-derived neurotrophic factor (GDNF) partially protected differentiated DA neurons against 6-OHDA-induced toxicity. These results suggest that undifferentiated and differentiated immortalized DA neurons can be a useful experimental model to study relative sensitivity to neurotoxins and neuroprotective agents that could have relevance to fetal and adult neurons.     

Neuropathology and behavioral impairments in Wistar rats with a 6-OHDA lesion in the substantia nigra compacta and exposure to a static magnetic field

Studies have sought to assess various potential neuroprotective therapeutics in Parkinson's disease. The aim of this study was to evaluate the effects of static magnetic field stimulation 14 days after a 6-Hydroxydopamine (6-OHDA) substantia nigra compacta (SNc) lesion on motor behavior, as assessed by the rotarod (RR) test and brain tissue morphology. Forty male Wistar rats were used and were divided into five groups: control group, sham group (SG), lesion group (LG), lesion north pole group (LNPG) and lesion south pole group (LSPG). In groups with magnetic stimulation, a 3200-gauss magnet was fixed to the skull. After the experiments, the animals were anesthetized for brain perfusion. Coronal sections of the SNc were stained with Nissl. The RR test showed a decrease in the time spent on the apparatus in the LG compared with all groups. The LNPG and LSPG had significant increases in the time spent when compared to the LG. A morphometric analysis revealed a significant reduction in the number of neurons in the LG, LNPG and LSPG in relation to the SG. There were a higher number of neurons in the LNPG and LSPG than the LG, and a higher number of neurons in the LSPG than the LNPG. We observed that the LG, LNPG and LSPG showed a higher number of glial cells than the SG, and the LNPG and LSPG showed a lower number of glial cells than the LG. Our results demonstrate a potential therapeutic use of static magnetic fields for the preservation of motor behavior and brain morphology in the SNc after 14 days with 6-OHDA lesion.     

Evaluation of Estrogen Neuroprotective Effect on Nigrostriatal Dopaminergic Neurons Following 6-Hydroxydopamine Injection into the Substantia Nigra Pars Compacta or the Medial Forebrain Bundle

Studies involving estrogen treatment of ovariectomized rats or mice have attributed to this hormone a neuroprotective effect on the substantia nigra pars compacta (SNpc) neurons. We investigated the effect of estradiol replacement in ovariectomized rats on the survival of dopaminergic mesencephalic cell and the integrity of their projections to the striatum after microinjections of 1 microg of 6-hydroxydopamine (6-OHDA) into the right SNpc or medial forebrain bundle (MFB). Estradiol replacement did not prevent the reduction either in the striatal concentrations of DA and metabolites or in the number of nigrostriatal dopaminergic neurons following lesion with 1 microg of 6-OHDA into the SNpc. Nevertheless, estradiol treatment reduced the decrease in striatal DA following injection of 1 microg of 6-OHDA into the MFB. Results suggest therefore that estrogen protect nigrostriatal dopaminergic neurons against a 6-OHDA injury to the MFB but not the SNpc. This may be due to the distinct degree of lesions promoted in these different rat models of Parkinson's disease.     

Basic Fibroblast Growth Factor Stimulation of Glial Cells Protects Dopamine Neurons from 6-Hydroxydopamine Toxicity: Involvement of the Glutathione System

Abstract: Neurotrophic factors have been shown to support the survival and promote the recovery of injured neurons both in vivo and in vitro. Here, we investigated whether glial cell line-derived neurotrophic factor (GDNF) and basic fibroblast growth factor (bFGF) could modify the damage to dopamine (DA) neurons in mesencephalic cultures caused by the neurotoxin 6-hydroxydopamine (6-OHDA). The data show that bFGF, but not GDNF, effectively protected DA neurons from 6-OHDA toxicity. Because bFGF is a glial mitogen, whereas GDNF is not, we tested whether glial cells participated in bFGF neuroprotection. Inhibition of glial cell proliferation completely prevented the protective effect of bFGF. Because oxidative events have been associated with 6-OHDA-induced damage, we examined the levels of glutathione (GSH) in control and bFGF-treated cultures. Cultures treated with bFGF had higher levels of GSH, which increased even further in response to 6-OHDA exposure. Control cultures failed to up-regulate GSH levels after 6-OHDA, suggesting a relationship between increased GSH levels and protection from 6-OHDA. Inhibition of glial cell proliferation prevented the rise in GSH in bFGF-treated cultures and abolished the increase after 6-OHDA treatment. Protection from 6-OHDA by bFGF was also diminished when GSH levels were decreased by the GSH synthesis inhibitor l -buthionine sulfoximine. Our study shows that stimulation of glial cells by bFGF allows the up-regulation of antioxidant defenses and supports cell survival during oxidative stress.     

Effects of 6-hydroxydopamine on brain and blood catecholamines, ammonia, and amino acids in rats

The effect of 6-hydroxydopamine (6-OHDA) upon brain and blood catecholamines, ammonia, and amino acids has been studied in rats subjected to increasing doses of the drug. Time dependent effects after injection have also been studied. Systemically injected 6-OHDA significantly, acutely reduced brain adrenaline (A), noradrenaline (NA), total catecholamines (TC), gamma-aminobutyric acid (GABA), and glutamic acid (Glu); concomitantly brain ammonia (NH3) increased. In blood, NA and TC were reduced and A and NH3 increased. The changes in brain monoamines are surprising since it has been reported that 6-OHDA does not cross the blood-brain barrier. We have proposed that these changes result from a general stress response or a reflex peripheral sympathetic response to falling blood pressure which in some manner communicates to the central nervous system. As the dose of 6-OHDA increased, brain NH3 increased and Glu decreased. A similar effect was seen from a single dose as the time after injection for sampling brain and blood constituents increased. Blood ammonia increases without change in Glu, glutamine, or asparagine. The source of NH3 may be from deamination of adenine nucleotide or catechols released from nerve terminals under the abnormal stimulus of 6-OHDA.     

Relationship between levels and uptake of serotonin and high affinity [3H]imipramine recognition sites in the rat brain

High affinity [3H]imipramine binding, endogenous levels of serotonin and noradrenaline, and serotonin uptake were determined in brain regions of rats with selective destruction of serotonergic neurons by 5,7-dihydroxytryptamine (5,7-DHT), of adrenergic neurons by 6-hydroxydopamine (6-OHDA), and of rats treated with reserpine. Neonatal treatment with 5,7-DHT resulted in a significant decrease of both serotonin levels and density (Bmax) of high affinity [3H]imipramine binding sites in the hippocampus. In contrast, an elevation of serotonin levels and an increase in Bmax of [3H]imipramine binding were noted in the pons--medulla region. No changes were observed in the noradrenaline content in either of these regions. Intracerebral 6-OHDA lesion produced a drastic suppression of noradrenaline levels in cerebral cortex but failed to alter the binding affinity (KD) or density (Bmax) of [3H]imipramine recognition sites. A single injection of reserpine (2.5 mg/kg) resulted in marked depletion of both serotonin (by 57%) and noradrenaline (by 86%) content and serotonin uptake (by 87%) in the cerebral cortex but had no significant influence of the parameters of high affinity [3H]imipramine binding in this brain region. The results suggest that high affinity [3H]imipramine binding in the brain is directly related to the integrity of serotonergic neurons but not to the magnitude of the uptake or the endogenous levels of the transmitter, and is not affected by damage to noradrenergic neurons or by low levels of noradrenaline.     

帕金森病大鼠中缝背核5-羟色胺能神经元电活动的变化

本实验采用玻璃微电极细胞外记录法,观察了帕金森病(Parkinson’s disease,PD)大鼠中缝背核(dorsal raphe nucleus, DRN)5-羟色胺(5-hydroxytryptamine,5-HT)能神经元电活动的变化。在大鼠右侧中脑黑质致密部内微量注射6-羟多巴胺(6- hydroxydopamine,6-OHDA)制作PD模型。结果显示,对照组和PD组大鼠DRN中5-HT能神经元的放电频率分别是(1．76±0．11)spikes／s(n=24)和(2．43±0．17)spikes／(n=21),PD组大鼠的放电频率显著高于对照组(P<0．001)。在对照组大鼠,92％(22／24)的神经元呈规则放电,8％(2／24)为爆发式放电;在PD组大鼠,具有规则、不规则和爆发式放电的神经元比例分别为9％(2／21)、43％(9／21)和48％(10／21),爆发式放电的5-HT能神经元比例明显高于对照组(P<0．001)。在对照组大鼠,DRN内局部注射5-HT1A拮抗剂WAY-100635(3μg/200nL)显著增加5-HT能神经元的放电频率而不影响其放电形式(n=19,P<0．002);而WAY-100635不改变PD组大鼠5-HT能神经元的放电频率和放电形式(n=17,P>0．05)。结果提示,用6-OHDA损毁黑质致密部造成的PD模型大鼠中神经元5-HT1A受体功能失调,并且DRN参与PD的病理生理学机制。     

Effect of benserazid and 6-hydroxydopamine on the development of the last larval instar of the house cricket, Acheta domestica L

The effect of benserazid and 6-OHDA on the duration of the last larval instar and development of wings with intact corpora allata and following allatectomy on Acheta domestica L. was studied. 6-OHDA failed to alter the duration of the last larval instar in the nymphs with intact corpora allata but prolonged it in the allatectomized crickets. Introduction of 6-OHDA in the second phase of the last larval instar caused a deformation of the wings both in allatectomized and intact crickets. Injection of benserazid to the nymphs of the last larval instar prolonged the duration of this phase only in the presence of the corpora allata. The effect of benserazid can be connected to elimination of central inhibition of the corpora allata involving dopaminergic neurons of the brain.     

EFFECTS OF 6-HYDROXYDOPAMINE ON CATECHOLAMINE CONTAINING NEURONES IN THE RAT BRAIN

After the intraventricular injection of 6-hydroxydopamine (6-OHDA), there was a long lasting reduction in the brain concentrations of noradrenaline (NA) and dopamine (DA). The brain concentration of NA was affected by lower doses of 6-OHDA than were required to deplete DA. A high dose of 6-OHDA which depleted the brain of NA and DA by 81 per cent and 66 per cent respectively, had no significant effect on brain concentrations of 5-hydroxytryptamine (5-HT) or γ-aminobutyric acid (GABA). The fall in catecholamines was accompanied by a long lasting reduction in the activities of tyrosine hydroxylase and DOPA decarboxylase in the hypothalamus and striatum, areas in the brain which are rich in catecholamine containing nerve endings. There was, however, no consistent effect on catechol-O-methyl transferase or monamine oxidase activity in these brain regions. The initial accumulation of [³H]NA into slices of the hypothalamus and striatum was markedly reduced 22–30 days after 6-OHDA treatment. These results are consistent with the evidence in the peripheral sympathetic nervous system that 6-OHDA causes a selective destruction of adrenergic nerve endings and suggest that this compound may have a similar destructive effect on catecholamine neurones in the CNS.     

Sympathetic and hyperthermic reactions by orexin A: role of cerebral catecholaminergic neurons

This experiment tested the effect of a lesion of cerebral catecholaminergic neurons on the sympathetic and thermogenic effects induced by an intracerebroventicular (icv) injection of orexin A. The firing rates of the sympathetic nerves to the interscapular brown adipose tissue (IBAT), along with IBAT, colonic temperatures and heart rate were monitored in urethane-anesthetized male Sprague-Dawley rats before an injection of orexin A (1.5 nmol) into the lateral cerebral ventricle and over a period of 150 min after the injection. Three days before the experiment, the rats were pre-treated with an icv injection of 6-hydroxydopamine (6-OHDA) or 6-OHDA plus desipramine or saline. The results show that orexin A increases the sympathetic firing rate, IBAT, colonic temperatures and heart rate in the rats pre-treated with saline. This increase is blocked by the pre-treatment with 6-OHDA alone or 6-OHDA plus desipramine. These findings indicate that cerebral catecholaminergic neurons (particularly the dopaminergic pathway) play a fundamental role in the complex reactions related to activation of the orexinergic system.     

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.