Degeneration of dopaminergic neurons in the substantia nigra of zitter mutant rat and protection by chronic intake of Vitamin E

Dopaminergic cell death in the ventral and dorsal tiers of substantia nigra pars copmacta (SNc) and their prevention by anti-oxidant diet was immunohistochemically studied in the zitter mutant rats, which are characterized by abnormal metabolism of superoxide. Similar to previous reports, the number of SNc neurons in Nissl-stained section decreased with age. Tyrosine hydroxylase (TH) immunohistochemistry demonstrated that the dopaminergic neurons in the ventral tier of SNc degenerated early, whereas the dorsal tier gradually degenerated with age. Thus, the ventral tier dopaminergic neurons are affected first, but the dorsal tier neurons do become impact by the zi/zi mutation. Following 9-month period after weaning, zitter rats supplemented with 500 mg d,l--tocophenol (VE(+))/kg diet exhibited a significant increased of surviving TH-immunoreactive neurons in both the tiers of SNc as compared with the zi/zi rats with control and VE(−) diets. These results suggest that VE supplement may slow the dopaminergic cell loss in zitter mutant rat, and further support that degeneration of the dopaminergic neurons in this mutant rat is caused by oxidant stress. Thus, the zitter rat may represent a good model for studying the dopaminergic cell death by superoxide species.     

Correlation between dopaminergic neurons, acetylcholinesterase and nicotinic acetylcholine receptors containing the alpha3- or alpha5-subunit in the rat substantia nigra

The aim of this study was to investigate the relationship between the cells possessing the alpha3 or alpha5 nicotinic acetylcholine receptor subunits and the enzyme acetylcholinesterase, with respect to tyrosine hydroxylase immunoreactive dopaminergic neurons in the rat substantia nigra. Most, but certainly not all, acetylcholinesterase immunoreactive cells were located in the pars compacta. In the substantia nigra pars compacta there were in turn two populations of acetylcholinesterase containing neurons: those that were tyrosine hydroxylase reactive and those that were not. Double label studies, that included an antibody immunoreactive against a common immunogen on alpha1 of muscle and alpha3 and alpha5 neuronal nicotinic acetylcholine receptor subunits, revealed that nearly all nicotinic receptor positive cells were also tyrosine hydroxylase neurons. However, a minority non-tyrosine hydroxylase population was alpha3- and/or alpha5-nAChR positive and these were always AChE-immunoreactive. In summary, there appears to be a close correlation between nicotinic receptors and acetylcholinesterase in the substantia nigra, irrespective of the transmitter phenotype in different neuronal subpopulations.     

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O₂/92% N₂) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.     

Activation of cultured rat hypothalamic dopaminergic neurons by long-term but not short-term treatment with prolactin

Pituitary prolactin (PRL) secretion is inhibited by hypothalamic GABAergic and dopaminergic (DAergic) systems. PRL, in turn, appears to be capable of activating these neurons, thus, providing for a negative feedback regulation. We have recently shown that cultured hypothalamic GABAergic- but not DAergic neurons respond to PRL with a rapid increase in intracellular free calcium. Here, we demonstrate that cultured hypothalamic DAergic neurons can be activated in terms of synthesis of dihydroxyphenylalanine (DOPA) by long-term PRL treatment. Short-term PRL treatment was ineffective. It is concluded that hypothalamic DAergic neurons are indeed capable of responding to PRL. However, their response differs from that of GABAergic neurons with respect to time scale and signal transduction. We suggest that the two types of hypothalamic cells are involved in separate feedback loops that provide for tonic and rapid regulation of pituitary PRL secretion, respectively.     

鱼藤酮损伤大鼠黑质至行为学及黑质多巴胺能神经元损伤

目的 探讨不同剂量鱼藤酮损伤大鼠黑质后行为学和酪氨酸羟化酶免疫活性细胞的变化.方法 采用大鼠黑质立体定位注射不同剂量鱼藤酮的方法,观察大鼠行为学变化,应用免疫组化染色检测黑质酪氨酸羟化酶(TH)免疫活性细胞的变化.结果 给鱼藤酮21d后,2.5μg/μl组大鼠行为测试记分2-4分为主,占91.3%,TH免疫活性细胞百分...     

Chronic exposure to aluminum reduces tyrosine hydroxylase expression in the substantia nigra and locomotor performance in rats

Aluminum (Al) is a neurotoxic agent that accumulates in the substantia nigra of patients affected by Parkinson's disease and in other cerebral areas of different neurodegenerative pathologies. Al has been associated with neuronal and glial dysfunctions, and neuronal changes have been suggested to affect several neurotransmitter systems including the dopaminergic system. The present study was designed to evaluate by means of immunohistochemistry using antibodies against tyrosine hydroxylase (TH; the rate-limiting enzyme of dopamine synthesis) the effects of chronic Al exposure (0, 3%) in drinking water during 4 months in adulthood or since intra-uterine age in the substantia nigra. Our results show a significant decrease in the number of cells labeled by the antibody against TH in rats treated with Al compared to controls. The TH-immunoreactive decrease following Al treatment is accentuated in the rat group treated since intrauterine age. In both treated groups, Al exposure induced a significant decrease of locomotor performance. Interestingly, as for TH-immunoreactivity, the decreased locomotor activity was also accentuated in the group treated since intrauterine age. The Al-induced TH alterations may be one of the causes of aluminum-induced neurotoxicity.     

Stereologic analysis of cell number and size during postnatal development in the rat substantia nigra

Parkinson's disease is characterized by age-related atrophy and loss of dopaminergic neurons within the compact portion of the substantia nigra (SNpc) projecting to neostriatum. Despite numerous studies using rodent models to examine mechanisms underlying this disorder, the fundamental question of whether development- or age-related changes occur in the rodent SNpc remains unanswered. The present study used a three-level, optical fractionator approach to estimate the number and size of SNpc neurons immunoreactive for tyrosine hydroxylase (TH) in eight young (2-month) and eight older (7-month) Sprague–Dawley rats. Following standard protocols for animal care and tissue harvesting, every eighth 60-μm section from a gapless coronal series was treated immunohistochemically for TH along with a thionin counterstain. Neither the ventral tegmental area nor the lateral part of the SN was included in the analysis. The total bilateral number of SNpc TH+ neurons (∼8000) was equivalent between groups, whereas mean TH+ neuronal volume decreased significantly in the older group (∼18%). In contrast, volume of the SNpc increased with age by 17%, as did volume of the entire brain (24%). TH+ cells in the SNpc were also significantly larger on the left versus right side of the brain. These data are consistent with the hypothesis that age-related volumetric expansion of the SNpc is accounted for by an increase in the ratio between neuropil and average neuron somal size during intermediate postnatal development.     

脑内炎症对黑质多巴胺能神经元的选择性损伤作用

目的 探讨脑内炎症反应对中脑黑质多巴胺能神经元的选择性变性作用.方法 健康SD雄性大鼠10只,随机分为实验组和对照组,实验组行脂多糖(LPS)右侧脑室定位注射,对照组注射生理盐水.注射后48周用免疫组织化学或组织化学法观察黑质多巴胺能、中缝核5-羟色胺能及基底核胆碱能3种不同类型神经元的变性及上述不同脑区小胶质细胞的激活情况.结果 免疫组织化学染色显示,LPS组在黑质、海马、纹状体、中缝核部位均可见到OX6阳性小胶质细胞,说明不同脑区均出现炎症反应.不同类型神经元染色结果显示,LPS组黑质多巴胺能神经元胞体变小、染色变浅、突起减少甚至消失,神经元数量比对照组减少40.1%(P<0.01);5-羟色胺能神经元及胆碱能神经元形态及数量均无明显改变.结论脑室注射LPS导致的脑内炎症反应可选择性引起黑质多巴胺能神经元变性损伤.     

GABAergic control of rat substantia nigra dopaminergic neurons: role of globus pallidus and substantia nigra pars reticulata

Dopaminergic neurons in vivo fire spontaneously in three distinct patterns or modes. It has previously been shown that the firing pattern of substantia nigra dopaminergic neurons can be differentially modulated by local application of GABA(A) and GABA(B) receptor antagonists. The GABA(A) antagonists, bicuculline or picrotoxin, greatly increase burst firing in dopaminergic neurons whereas GABA(B) antagonists cause a modest shift away from burst firing towards pacemaker-like firing. The three principal GABAergic inputs to nigral dopaminergic neurons arise from striatum, globus pallidus and from the axon collaterals of nigral pars reticulata projection neurons, each of which appear to act in vivo primarily on GABA(A) receptors (see preceding paper). In this study we attempted to determine on which afferent pathway(s) GABA(A) antagonists were acting to cause burst firing. Substantia nigra dopaminergic neurons were studied by single unit extracellular recordings in urethane anesthetized rats during pharmacologically induced inhibition and excitation of globus pallidus. Muscimol-induced inhibition of pallidal neurons produced an increase in the regularity of firing of nigral dopaminergic neurons together with a slight decrease in firing rate. Bicuculline-induced excitation of globus pallidus neurons produced a marked increase in burst firing together with a modest increase in firing rate. These changes in firing rate were in the opposite direction to what would be expected for a monosynaptic GABAergic pallidonigral input. Examination of the response of pars reticulata GABAergic neurons to similar manipulations of globus pallidus revealed that the firing rates of these neurons were much more sensitive to changes in globus pallidus neuron firing rate than dopaminergic neurons and that they responded in the opposite direction. Pallidal inhibition produced a dramatic increase in the firing rate of pars reticulata GABAergic neurons while pallidal excitation suppressed the spontaneous activity of pars reticulata GABAergic neurons. These data suggest that globus pallidus exerts significant control over the firing rate and pattern of substantia nigra dopaminergic neurons through a disynaptic pathway involving nigral pars reticulata GABAergic neurons and that at least one important way in which local application of bicuculline induces burst firing of dopaminergic neurons is by disinhibition of this tonic inhibitory input.     

Meloxicam reduces lipopolysaccharide-induced degeneration of dopaminergic neurons in the rat substantia nigra pars compacta

Inflammation is believed to play an important role in the etiology and pathogenesis of Parkinson's disease (PD). However, experimental and epidemiological evidences from various non-steroidal anti-inflammatory drugs, including cyclooxygenase-2 (COX-2) inhibitors, seem contradictive. Using the intranigral lipopolysaccharide (LPS) rat model, we show that meloxicam, a preferential COX-2 inhibitor, diminishes the activation of OX-42-immunoreactive (ir) microglia and reduces the loss of tyrosine hydroxylase (TH)-ir dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) that is normally induced by exposure to LPS. Double-labelling immunohistochemistry identified that activated microglia rather than intact resting microglia are the main intracellular venues for COX-2 expression. These findings suggest that inhibition of COX-2 activity in activated microglial cells may be potentially neuroprotective for DA neurons in the SNpc.     

尼古丁对PD大鼠黑质多巴胺能神经元变性的影响

目的 探讨尼古丁对帕金森病(PD)大鼠黑质多巴胺能神经元变性的影响及其机制. 方法 45只大鼠随机分为PBS对照组(CON)、生理盐水+ 脂多糖(NS)组、尼古丁+脂多糖(NIC)组,每组15只.黑质内立体定向注射脂多糖(LPS)或PBS后24h,免疫印迹法检测黑质诱导性一氧化氮合酶(iNOS)蛋白表达变化;黑质注射药物后14d,采用免疫组织化学法观察大鼠黑质酪氨酸羟化酶(TH)阳性神经元数量及OX-42阳性细胞形态学变化,RT-PCR及免疫印迹检测黑质TH mRNA及TH蛋白的表达水平. 结果 与CON组相比,NS组大鼠黑质iNOS表达明显增多,TH阳性神经元、TH mRNA及TH蛋白明显减少,小胶质细胞大多呈胞体大突起短粗的形态;NIC组黑质iNOS表达明显少于NS组,黑质TH阳性神经元、TH mRNA及TH蛋白表达较NS组明显增多,大部分小胶质细胞呈胞体小,突起细长的形态. 结论 尼古丁可以减轻LPS介导的多巴胺能神经元变性,对多巴胺能神经元有保护作用,其保护机制与抑制小胶质细胞激活、减少iNOS的表达有关.     

Anatomical and functional compartmentalization of the subparafascicular thalamic nucleus in the rat

Summary The localization and the transmitter phenotype of subparafascicular thalamic nucleus (Spf) neurons projecting to the inferior colliculus (IC) and to the spinal cord (Sp) were studied by using a retrograde fluorescent double labeling technique, and a combined technique of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH) and glutamate decarboxylase (GAD). The cell population of Spf-IC neurons was totally differentiated from that of Spf-Sp neurons which have been reported to be dopaminergic. The former were densely distributed, small to medium sized cells and localized in the central portion of the Spf, while the latter were sparsely distributed, large cells and localized in the marginal portion of the Spf. Spf-IC neurons were completely devoid of TH immunoreactivity and, instead, approximately half of them showed GAD immunoreactivity. From these findings, it is concluded that the Spf is distinctly compartmentalized by the presence at least two separate neuronal subpopulations, which are distinguishable in terms of their cell size, distribution patterns, transmitter phenotypes and trajectories.Abbreviations BC brachium conjunctivum - FG Fluoro-gold - FITC Fluorescein isothiocyanate - FR fasciculus retroflexus - GAD glutamate decarboxylase - Hb habenula - IC inferior colliculus - PI Propidium iodide - Sp spinal cord - Spf sub-parafascicular thalamic nucleus - TH tyrosine hydroxylase     

Increases in TH immunoreactivity, neuromelanin and degeneration in the substantia nigra of middle aged mice

The dopaminergic (DArgic) neurons in the substantia nigra (SN) are particularly vulnerable to oxidative stress and during aging. The present study was undertaken in order to determine whether aging is associated with changes in the DA synthesizing enzyme tyrosine hydroxylase (TH) as early as middle age by comparing 7- and 50-week-old mice. Quantitative analysis, performed by measuring the density of TH-immunopositive neurons, revealed that in the older animals, the number of DArgic neurons was decreased by 10% while TH immunodensity was 24 +/- 3% higher compared to the younger animals. Based on Masson-Fontana staining for neuromelanin (NM), the number of NM-containing neurons in the SN and the volume of NM per NM-positive neurons in the older animals were 5- and 11.6 +/- 0.1-fold higher, respectively. The silver stain-positive fibers, indicative of degeneration, were higher in the SN and striatum of the older animals, with the optical density 3.3 +/- 0.1- and 5.4 +/- 0.2-fold of the younger animals. The present study demonstrates that aging is associated with changes in the DA synthesizing enzyme TH as early as middle age and that this is associated with dramatic increases in the number of NM-containing neurons, volume of NM per cell, and degeneration.     

乙醇处理与大鼠脑内多巴胺能体系的关系

目的观察乙醇处理大鼠脑内酪氨酸羟化酶(TH)和多巴胺转运体(DAT)的表达变化,判断乙醇对脑内多巴胺(DA)能体系的影响。方法选取Wistar大鼠60只,随机分为对照组和乙醇处理组,每组30只,乙醇处理组大鼠以20%乙醇代替饮水饲养6个月;利用免疫组织化学、流式细胞术及免疫印迹等方法,分析乙醇处理大鼠有关脑区TH和DAT的表达改变。结果1.免疫细胞化学法研究发现,乙醇处理组大鼠脑内黑质(SN)-腹侧被盖区(VTA)、尾壳核(CPu)和伏核(NACC)TH的灰度值明显低于对照组(P0.05);尾壳核和伏核DAT的灰度值明显低于对照组(P0.05)。2.流式细胞术检测发现,乙醇处理组大鼠中脑TH的表达量明显高于对照组(P0.05)。3.免疫印迹检测发现,乙醇处理组大鼠所观察脑区TH和DAT与β-actin条带相对吸光度比值明显高于对照组(P0.05)。结论乙醇处理增加大鼠脑内TH和DAT的表达。     

Dual immunofluorescence study of citrullinated proteins in Parkinson diseased substantia nigra

Deimination is a post-translational modification of proteins in which selected arginine amino acids are enzymatically converted to citrullines. Using dual-color immunofluorescence and an established monoclonal antibody (F95) against peptidyl-citrulline moieties, the present study is the first to compare immunohistochemical staining patterns for deiminated proteins in human substantia nigra (SN) from patients with Parkinson disease (PD) versus similar control specimens supplied by the Harvard Brain Bank. In control SN sections, many tyrosine hydroxylase (TH)-immunoreactive dopamine neurons were seen surrounded either by small fibers immunoreactive for deiminated proteins, or large reactive astrocytes, co-localized with glial fibrillary acidic protein (GFAP). However, in SN specimens from PD patients, immunoreactivity for deiminated proteins was also demonstrated within the cytoplasm of many surviving dopamine neurons that were also immunoreactive for TH, but this staining was not specifically restricted to Lewy bodies. Although the identity of neuronal deiminated proteins in these SN dopamine neurons is unknown, the present study provides evidence that the anatomical expression of these proteins in PD is altered and thus suggests that deimination may be involved in the pathophysiology of this disease.     

Inhibitory substantia nigra inputs to the pedunculopontine neurons

Summary Responses of 43 pedunculopontine area (PPN area) neurons to electrical stimulation of the substantia nigra (SN) were studied in anesthetized rats. An intracellular recording technique was used to demonstrate that SN stimulation evoked hyperpolarizing potentials, which were identified by intracellular injections as inhibitory postsynaptic potentials (IPSPs). These IPSPs were often followed by a rebound depolarization that originates several spike potentials. These IPSPs were characterized as monosynaptic, with latencies varying from 1.0 to 8.5 ms. Similar results were observed in some animals with chronic unilateral coronal lesion just rostral to subthalamic nucleus (STH), which severed the rostral afferents. PPN area neurons were also antidromically activated by SN stimulation. Two PPN area projection neurons were clearly identified. Mean latency of one group was 0.71 ms; mean latency of the second group was 5.16 ms. The morphological analysis of a neuron inhibited by SN stimulation and labeled with horseradish peroxidase (HRP) demonstrated that the soma was fusiform in shape, with the axon originating in the soma and collaterals and a large dendritic field extending in the ventrodorsalis direction. The results indicate that the PPN area is reciprocally connected with the SN, which elicits an inhibitory effect on PPN area neurons.     

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured catecholaminergic neurons in a chemically defined medium from fetal and postnatal rat midbrains

Interleukin-6 (IL-6, human recombinant) promoted the survival of catecholaminergic neurons from fetal and postnatal rat midbrains as assessed by an immunohistochemical staining for tyrosine hydroxylase (TH) in culture using a chemically defined medium. The maximal dose of IL-6 for the cell survival of postnatal P15 rat mesencephalic TH-positive neurons in culture for 7 days was 50 ng/ml. The survival-promoting effects on P15 cultures were observed both in high- and low-density cultures. The survival effect of IL-6 on the cultured P15 TH-positive neurons was significant for only 4-15 days in vitro. However, the viable number of TH-positive neurons with IL-6 was less than that of the control at early points in the culture process (1-2 days in vitro). Continuous presentation of IL-6 was required for promoting survival. The optimal dose of IL-6 for the survival of fetal E16 midbrain TH-positive neurons was 5 ng/ml, and the survival promoting effect was less than that for the P15 cultures. The maximal dose of IL-6 for the survival of P2 TH-positive neurons was 5 ng/ml and that of P8 was 50 ng/ml, indicating that the response of rat mesencephalic TH-positive neurons to IL-6 changes during the first postnatal week.     

天麻对帕金森病大鼠黑质凋亡细胞表达及酪氨酸羟化酶阳性神经元的影响

目的研究和探讨帕金森病（PD）的发病机理及天麻防治PD的作用机制。方法采用6-羟基多巴胺（6-OHDA）大鼠模型,通过免疫组化技术观察天麻对PD大鼠黑质酪氮酸羟化酶阳性（TH^＋）神经元及Caspase-3阳性（Caspase-3^＋）神经元表达水平的影响。结果PD大鼠脑组织黑质致密部（SNpc）及腹侧被盖区（VTA）TH^＋神经元表达水平组间比较差异有统计学意义（F值分别为13．29、5．04,P〈0．01）。与正常组大鼠脑组织SNpc和VTA区TH^＋神经元表达水平[（32．13±4．84）,（30．23±3．21）]比较,模型组大鼠TH^＋神经元表达水平[（18．89±3．73）和（24．20±6．35）]明显下降（P〈0．01）;经天麻和美多巴治疗后TH^＋神经元丢失减少,天麻小剂量组TH^＋神经元表达水平分别为（26．24±3．06）和（26．31±6．1）,中剂量组分别为（22．44±3．81）和（26．91±6．28）;大剂量绀分别为（20．95±4．71）和（27．3±7．1）;美多巴组分别为（27．89±4．56）和（25．974±5．14）;其中,天麻小剂量组和美多巴组SNpc区TH^＋神经元表达水平和模型组比较差异有统计学意义（P〈0．05）。PD大鼠脑组织SNpc及VTA区Caspase-3阳性细胞表达水平组间比较差异有统计学意义（F值分别为6．09、5．53,P〈0．01）。与正常组Caspase-3阳性细胞表达水平[（9．83±3．03）,（7．04±1．76）]比较,模型组火鼠Caspase-3阳性细胞表达水平[（14．53±2．33）和（12．84±2．58）]明显升高（P〈0．01）;经天麻和美多巴治疗后Caspase-3阳性细胞表达水平下降,天麻小剂量组分别为（10．68±1．83）和（7．72±1．92）,中剂量组分别为（11．29±2．8）和（10．38±3．79）;大剂量组分别为（11．89±2．97）和（11．37±1．86）;美多巴组分别为（9．99±3．3）和（10．69±3．11）;其中,美多巴组SNpc区和天麻小剂量组VTA区Caspase-3阳性细胞表达水平和模型组比较差异有统计学意义（P〈0．05）。天麻和美多巴治疗各组TH^＋神经元表达与Caspase-3^＋神经元表达呈相反趋势。结论细胞凋亡可能是PD大鼠中腑多巴胺神经元丢失的主要方式。天麻可通过调节Caspase-3减少细胞凋亡,其中,小剂量天麻对治疗PD具有较好的作用。本研究结果提示天麻可不同程度地保护多巴胺神经元。     

Distribution of dopaminergic cell bodies in the median raphe nucleus of the rat brain

An increasing amount of data suggests that a dysfunction in dopamine (DA) neurotransmission is involved in the pathophysiology of various neurological and psychiatric disorders. With this in mind, the distribution and connectivity of the dopaminergic system in the rat brain has been studied extensively. So far, little is known about the distribution of DA containing neurons in the median raphe nucleus (MnR). This nucleus is mainly defined by a large population of serotonin containing neurons. Using quantitative immunohistochemistry, we observed the presence of a small number of DA containing neurons in the rat MnR, which was in contrast to a previous report.     

Isthmic origin of neurons of the rat substantia nigra

This study provides new data on the time of origin, the generation site and the migration route of the young neurons of the substantia nigra of the rat during embryogenesis. The neurons of the substantia nigra are generated on day 12, 13, 14 and 15 of gestation. They settle following a light spatiotemporal rostrocaudal gradient from day 12 to 15. The neurons of the substantia nigra are generated at two different points of the basal plate at the level of the fovea isthmi (meso-isthmic junction) and migrate in radial pattern as two definite streams toward the ventral mesencephalon. From this point they move rostralwards along the surface towards their final site.The main findings of this work are the disclosure that the neurons of the substantia nigra are generated in the region of the isthmus rhombencephali and that its cells do not migrate between existing cells of the mesencephalic tegmentum but first migrate ventralwards in a radial pattern and then rostrally towards their definite site. Numerous neurons of the basal mesencephalon and of the midline structures of the caudal mesencephalon are apparently derived from the region of the fovea isthmi.