Development of GABAergic neurons from the ventricular zone in the superior colliculus of the mouse

The superior colliculus (SC) is a layered structure in the midbrain and is particularly rich in gamma-aminobutyric acid (GABA). The present investigation aimed to determine whether the development of GABAergic neurons in the SC is common to that of the neocortex in which they are produced in a distinct area called the ganglionic eminence and are transported by tangential migration. A green fluorescent protein (GFP) knock-in mouse was used in which a GFP gene was introduced into the gene for glutamic acid decarboxylase (GAD) 67 and all GABAergic neurons were fluorescent. At embryonic day (E) 11-14, GFP-positive cells increased strikingly. They were spindle-shaped with processes at both poles and oriented radially between the ventricular and pial surface, together with other GFP-negative cells. After the cutting of the embryonic SC, GFP-positive cells accumulated on one side of the injury as expected from their radial but not tangential migration. In the living slice preparations GFP-positive cells migrated radially during the observation. These results indicate that tangential migration of GABAergic neurons as observed in the neocortex is not applicable and that radial migration from the underlying ventricular zone is predominant in the SC. At E12-13, bundles of commissural GFP-positive fibers which appeared to originate outside the SC were distributed at the superficial layer. These superficial fibers were no longer observed at the later stages.     

Morphological development and neurochemical differentiation of cerebellar inhibitory interneurons in microexplant cultures

The cerebellar cortex comprises a rather limited variety of interneurons, prominently among them inhibitory basket and stellate cells and Golgi neurons. To identify mechanisms subserving the positioning, morphogenesis, and neurochemical maturation of these inhibitory interneurons, we analyzed their development in primary microexplant cultures of the early postnatal cerebellar cortex. These provide a well-defined, patterned lattice within which the development of individual cells is readily accessible to experimental manipulation and observation. Pax-2-positive precursors of inhibitory interneurons were found to effectively segregate from granule cell perikarya. They emigrate from the core explant and avoid the vicinity of granule cells, which also emigrate and aggregate into small clusters around the explant proper. This contrasts with the behavior of Purkinje neurons, which remain within the explant proper. During migration, a subset of Pax-2-positive cells gradually acquires a GABAergic phenotype, and subsequently also expresses the type 2 metabotropic receptor for glutamate, or parvalbumin, markers for Golgi neurons and basket or stellate cells, respectively. The latter eventually orient their dendrites such that they take a preferentially perpendicular orientation relative to granule cell axons. Both the neurochemical maturation of basket/stellate cells and the specific orientation of their dendrites are independent of their continuous contact with radially oriented glia or Purkinje cell dendrites projecting from the core explant. Numbers of parvalbumin-positive basket/stellate cells and the prevalence of glutamate-positive neurites, which form a dense network preferentially within cell clusters containing granule cell perikarya and their dendrites, are subject to regulation by chronic depolarization. In contrast, brain-derived neurotrophic factor results in a drastic decrease of numbers of basket/stellate cells. These findings document that granule cell axons (parallel fibers) are the major determinant of basket/stellate cell dendritic orientation. They also show that the neurochemical maturation of cerebellar interneurons is sensitive to regulation by activity and neurotrophic factors.     

小鼠大脑新皮质片层化形成过程和细胞周期变化

目的 探讨小鼠大脑新皮质片层化的组织发生过程和细胞周期的关系,对有丝分裂后神经元在迁移中的细胞周期变化、神经细胞的增殖、神经元的迁移进行观察.方法 各日龄共计200只小鼠,应用免疫荧光法、5′-溴脱氧尿嘧啶核苷(BrdU)检测和DiI标记技术对胚胎期和出生后小鼠的大脑皮质进行形态学观察,对皮质BrdU和Cyclin D1阳性细胞密度进行测量.结果 皮质板最早在胚龄15d(E15)时形成,小鼠大脑新皮质深层(第Ⅵ～Ⅴ层)片层化进程开始于生后0 d(P0),皮质浅层(第Ⅳ～Ⅱ层)的片层化趋势开始于P5,P7时6层结构完全形成,但未呈现片层化特点,P14时小鼠大脑新皮质片层化完全形成,P30时片层化结构趋于稳定.在大脑新皮质片层化过程中,锥体细胞在E17时呈椭圆形,树突有小分支,在P15时发育成熟,呈锥形并有复杂的顶树突和基树突.BrdU检测发现,室管层和室管层下区有大量增殖的干细胞,在此期间由BrdU阳性细胞增殖生成的有丝分裂后神经元可以迁移到大脑新皮质;P0至P30,迁移到皮质板的有丝分裂后神经元逐渐减少.利用G1期特异性标记物Cyclin D1对有丝分裂后神经元的细胞周期进行分析发现,有丝分裂后神经元处于G1期,它们一旦定居到皮质板将退出细胞周期.新皮质中Cyclin D1阳性细胞数量呈抛物线变化,在P12达到峰值,P30后在皮质板只能发现少量的Cyclin D1阳性细胞.结论 小鼠大脑新皮质片层化过程经历了细胞增殖、分化与迁移,同时伴随着皮质板锥体细胞的成熟.神经细胞的增殖和迁移主要发生在胚胎期和生后早期,迁移的细胞主要处于G1期.有丝分裂后神经元的分化过程实际上是G1期到G0期的过渡,一旦在皮质板定居下来,有丝分裂后神经元将退出细胞周期,进入G0期.     

Spatial and temporal distribution patterns of enkephalinergic neurons in adult and developing retinas of the preproenkephalin-green fluorescent protein transgenic mouse

Enkephalin (ENK) peptides are present in the retina of several vertebrate species and play a crucial role in establishing specific circuits during retinal development. However, there is no information available concerning the development of ENKergic neurons in the mouse retina. To address this question, we used preproenkephalin-enhanced green fluorescent protein (GFP) transgenic mice, in which ENKergic neurons are revealed by GFP. Our results showed that most GFP-positive cells were located in the proximal part of the inner nuclear layer with a scattering of GFP-immunoreactive cells in the ganglion cell layer (GCL) in the adult retina. Double immunostaining with syntaxin indicates that GFP expression was restricted to a population of amacrine cells. The proportions of glycine transporter-1 and γ-aminobutyric acid-positive cells among ENKergic neurons were 57.3 ± 2.4% and 10.1 ± 1.8%, respectively. We then injected retrograde tracer into the superior colliculus and observed that none of the ENKergic neurons in the GCL were retrogradely labeled with the tracer. GFP-positive cells were first observed at embryonic day (E) 15 in the inner neuroblastic layer at only very low levels, which gradually increased until E18. After birth, there was a steep rise in GFP expression levels, reaching maximal activity by postnatal day (P) 7. The distribution and intensity of GFP-positive cells at P15 were similar to those of adult retina. It was found that immunoreactive processes in the inner plexiform layer formed strongly stained patches. The present results provide detailed morphological evidence of the cell type and spatial and temporal distribution of ENKergic neurons in the retina.     

Brain-derived neurotrophic factor modulates dendritic morphology of cerebellar basket and stellate cells: an in vitro study

The dendrites of cerebellar basket/stellate cells show a highly stereotyped orientation relative to granule cell axons (parallel fibers) and Purkinje cell dendrites. This specific morphology is acquired during the early postnatal phase of cerebellar development, when basket/stellate cells become synaptically integrated with Purkinje neurons and granule cells. In the present study, we used primary cerebellar cultures to test how the spatial arrangement of granule cell axons affects basket/stellate cell dendritic morphology. In addition, we sought to determine whether active signals as might be provided by granule cells, i.e. synaptic input and the neurotrophin, brain-derived neurotrophic factor, affect basket/stellate cell development. Our results confirm the critical role of parallel fiber orientation for basket/stellate dendritic morphogenesis. Moreover, we found that both electrical activity and brain-derived neurotrophic factor increased basket and stellate cell dendritic arborization.Together with previously published findings, our data led to the conclusion that both structural cues and active interneuronal signaling collaborate to bring about the precise morphogenesis of cerebellar basket/stellate cells. The distinct responses of various cerebellar phenotypes towards the morphogenetic effects of brain-derived neurotrophic factor suggest that this neurotrophin, within the developing cerebellum, enhances synaptic connectivity by concerting the formation of appropriate pre- and postsynaptic structures.     

Coordinated developmental recruitment of latent fast spiking interneurons in layer IV barrel cortex

Feedforward inhibitory GABAergic transmission is critical for mature cortical circuit function; in the neonate, however, GABA is depolarizing and believed to have a different role. Here we show that the GABAA receptor-mediated conductance is depolarizing in excitatory (stellate) cells in neonatal (postnatal day [P]3-5) layer IV barrel cortex, but GABAergic transmission at this age is not engaged by thalamocortical input in the feedforward circuit and has no detectable circuit function. However, recruitment occurs at P6-7 as a result of coordinated increases in thalamic drive to fast-spiking interneurons, fast-spiking interneuron-stellate cell connectivity and hyperpolarization of the GABAA receptor-mediated response. Thus, GABAergic circuits are not engaged by thalamocortical input in the neonate, but are poised for a remarkably coordinated development of feedforward inhibition at the end of the first postnatal week, which has profound effects on circuit function at this critical time in development.     

GAD67-GFP基因敲入小鼠黑质网状部神经元GABA与氯离子转运体KCC2和NKCC1的共存

为了观察谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠黑质网状部(SNr)内,表达GFP的GABA能神经元与一对功能相反的Cl-共转运体(K+-Cl-cotransporter2,KCC2;Na+-K+-Cl-cotransporter1,NKCC1)的共存情况,本研究分别运用原位分子杂交与免疫组织化学相结合以及GFP与KCC2或NKCC1免疫荧光染色相结合的双重标记方法,在光学显微镜和激光共聚焦显微镜下同时进行观察。结果显示:(1)SNr内95%以上的GFP阳性神经元同时表达KCC2 mRNA,而50%表达KCC2 mRNA的阳性神经元呈GFP阳性;(2)SNr内80%以上的GFP阳性神经元同时表达NKCC1 mRNA,约35%表达NKCC1 mRNA的阳性神经元呈GFP阳性;(3)SNr内90%以上的GFP阳性神经元同时表达KCC2,双标神经元约占KCC2阳性神经元的50.5%;(4)SNr内80.5%以上的GFP阳性神经元同时表达NKCC1,双标神经元约占NKCC1阳性神经元的42.5%。以上结果表明,SNr内表达GFP的GABA能神经元大部分与KCC2和NKCC1共存,提示氯离子共转运体可能对SNr内GABA能神经元起重要的调控作用。     

小鼠放射状胶质细胞和小脑皮质的发育

目的探讨小鼠小脑皮质发育过程中放射状胶质细胞的分化。方法应用免疫荧光及5-溴脱氧尿嘧啶核苷(BrdU)检测技术,标记小鼠胚胎8d至生后180d小脑(57例,分为19组,每组3只)的神经干细胞、放射状胶质细胞、普肯耶细胞及颗粒细胞。结果放射状胶质细胞于胚胎13d的神经上皮出现,尔后该细胞分化为各种神经元和贝格曼胶质细胞,并在小脑皮质层状结构的形成中起着重要作用。结论放射状胶质细胞来源于神经上皮细胞,是神经细胞和神经胶质细胞的前体细胞。在小脑皮质的发育过程中,放射状胶质细胞能分化为普肯耶细胞和颗粒细胞,并为神经细胞的迁移提供路径和支架。     

Immunogold demonstration of GABA in synaptic terminals of intracellularly recorded, horseradish peroxidase-filled basket cells and clutch cells in the cat's visual cortex

To identify the putative transmitter of large basket and clutch cells in the cat's visual cortex, an antiserum raised against GABA coupled to bovine serum albumen by glutaraldehyde and a postembedding, electron microscopic immunogold procedure were used. Two basket and four clutch cells were revealed by intracellular injection of horseradish peroxidase. They were identified on the basis of the distribution of their processes and their synaptic connections. Large basket cells terminate mainly in layer III, while clutch cells which have a more restricted axon, terminate mainly in layer IV. Both types of neuron have a small radial projection. They establish type II synaptic contacts and about 20-30% of their synapses are made with the somata of other neurons, the rest with dendrites and dendritic spines. Altogether 112 identified, HRP-filled boutons, the dendrites of three clutch cells and myelinated axons of both basket and clutch cells were tested for the presence of GABA. They were all immunopositive. The postsynaptic neurons received synapses from numerous other GABA-positive boutons in addition to the horseradish peroxidase-filled ones. Dendritic spines that received a synapse from a GABA-positive basket or clutch cell bouton also received a type I synaptic contact from a GABA-negative bouton. A few of the postsynaptic dendrites, but none of the postsynaptic somata, were immunoreactive for GABA. The fine structural characteristics of the majority of postsynaptic targets suggested that they were pyramidal and spiny stellate cells. These results provide direct evidence for the presence of immunoreactive GABA in identified basket and clutch cells and strongly suggest that GABA is a neurotransmitter at their synapses. The laminar distribution of the synaptic terminals of basket and clutch cells demonstrates that some GABAergic neurons with similar target specificity segregate into different laminae, and that the same GABAergic cells can take part in both horizontal and radial interactions.     

Distribution of mGluR1alpha and SMI 311 immunoreactive Lugaro cells in the kitten cerebellum

The Lugaro cell is a feedback interneuron of the cerebellar cortex, recognizable by its characteristic morphology. Postnatal neuronal migration to the cortex has been described for several cerebellar interneurons. Since in our previous studies we observed Lugaro-like cells (LCs) in the white matter (WM) and internal granular layer (IGL) of the cerebellum of young cats, we assumed that a proportion of these cells migrate also postnatally to their destination. In the present study using and immunostaining for the metabotropic glutamate receptor mGluR1alpha and neurofilament protein SMI 311 the number and spatial distribution of LCs at different postnatal days were investigated. We found that the number and distribution of both mGluR1a-immunoreactive (ir) and of SMI 311-ir LCs changed with age in the developing cerebellar cortex of kittens: developing LCs express mGluR1alpha already in the newborn, while expression of SMI 311-ir in LCs appears only about a week later. At postnatal day 1 (P1) relatively few mGluR1-ir LCs were detected in the WM and at the border of WM and IGL. Later, their number increased sharply until P15 (6-7 fold) and decreased continuously between P15 and P135. SMI 311-ir LCs were not present at P1 and even at P8 only a few were observed in the WM or in infraganglionic positions. Their number increased gradually (12-14 fold) until adulthood when their number was stabilized at 8.000-10.000/cerebellum. At the same time the number of probably ectopic SMI 311-ir LCs decreased with age: at P22 about one third of them was found in "ectopic" position, whereas in the adult cat only about 10-12% of LCs's was either in the WM or scattered in the whole depth of the granular layer. These results suggest that: (1) most LCs appear in the cerebellar cortex postnatally; and (2) postnatal migration and incorporation of LCs to the cortex is a much longer process than previously expected, occurring even after the cytoarchitectonic built-up (about P65-P70 in cat) of the cerebellum.     

Age-dependent and selective binding of beta-bungarotoxin to GABAergic neurons in the rat cerebellum

beta-Bungarotoxin (BuTx)-binding cells were immunocytochemically examined in the developing rat cerebellum. The tissue was incubated with BuTx and then immunostained with antiserum against its toxoid. On postnatal day 6, only Golgi cells were positive for immunoreaction. Immunoreactive Golgi cells were reduced in number on day 15 and disappeared on day 25. On day 15, Purkinje cells were strongly stained, while some basket and stellate cells stained weakly. On day 25 and in adult, basket and stellate cells were more immunoreactive than Purkinje cells. Thus, age-dependent and selective binding of BuTx was restricted to gamma-aminobutyric acid (GABA)ergic neurons.     

小鼠脊髓神经元内脑啡肽与1型囊泡膜谷氨酸转运体的共存

目的:以PPE-GFP转基因小鼠为研究工具,观察绿色荧光蛋白(GFP)阳性的脑啡肽(ENK)能神经元与1型囊泡膜谷氨酸转运体(VGLUT1)在脊髓的分布及共存情况。方法:利用免疫组织化学和原位杂交双标染色的方法。结果:GFP标记的ENK能神经元主要位于脊髓背角,在I-ⅡI层最为密集,背角深层内侧部及中央管周围呈中等密度分布,散在分布于前角。VGLUT1 mRNA阳性细胞广泛分布在脊髓各层。GFP/VGLUT1双标细胞主要分布在脊髓背角,I-ⅡI层双标细胞占GFP阳性细胞的22.95±1.10%,占VGLUT1阳性细胞的27.91±2.42%;IV-VI层中21.49±4.99%GFP阳性细胞表达VGLUT1,10.35±2.81%VGLUT1阳性细胞表达GFP;前角双标细胞占VGLUT1阳性细胞的1.07±0.37%,占GFP阳性细胞的32.08±13.15%。结论:双标结果表明脊髓内部分ENK能神经元表达1型囊泡膜谷氨酸转运体,推测ENK能神经元可能通过调控谷氨酸的释放发挥感觉信息调控作用。     

Mitotic activity and specification of fibrocyte subtypes in the developing rat cochlear lateral wall

Spiral ligament fibrocytes (SLFs) in the mammalian cochlear lateral wall participate in K⁺ recycling; they are classified into five subtypes based on their morphology, distribution, and function. Regeneration of SLFs is a potential therapeutic strategy for correcting several types of hearing loss, prompting us to investigate how SLF subtypes are established during development. We compared transitional SLF-type marker expression with mitotic activity to evaluate proliferation-differentiation relationships in SLFs from postnatal rat cochleae. I.p. injection of 5-bromo-2′-deoxyuridine (BrdU) demonstrated that the overall mitotic activity of SLFs decreased significantly between postnatal day 7 (P7) and P10. For all developmental periods, BrdU incorporation was weakest in the area where type I SLFs reside. The onset of expression of markers for type II/IV SLFs followed the reduced mitotic activity of the cells, whereas that of aquaporin-1, a marker for type III SLFs, was already detectable at P7, when the type III SLFs were still proliferating vigorously. Distribution of BrdU⁺ cells increased in the area of type I SLFs between P7 and P10, suggesting migration of SLFs from adjacent areas. We conclude that the time course of development of SLFs is subtype-specific.     

Medial septal and median raphe innervation of vasoactive intestinal polypeptide-containing interneurons in the hippocampus

Vasoactive intestinal polypeptide-immunoreactive interneurons are known to form three anatomically and neurochemically well-characterized neuron populations in the hippocampus. Two of these establish synaptic contacts selectively with other GABAergic cells (interneuron-selective cells), whereas the third type innervates pyramidal cell bodies and proximal dendrites like a conventional basket cell. Our aim was to examine which of the vasoactive intestinal polypeptide-containing interneuron populations are among the targets of GABAergic septohippocampal and serotonergic raphe-hippocampal pathways. Anterograde tracing with Phaseolus vulgaris leucoagglutinin combined with double immunocytochemistry for vasoactive intestinal polypeptide was used at the light and electron microscopic levels. Our results show that both interneuron-selective cells and vasoactive intestinal polypeptide-containing basket cells receive synaptic input from the medial septum and median raphe nucleus. The GABAergic component of the septohippocampal pathway establishes multiple contacts on both cell types. In the case of the raphe-hippocampal projection, single or double contacts were more frequent on vasoactive intestinal polypeptide-positive interneuron selective cells (76%), whereas multiple contacts predominated on basket cells (83%). The extrinsic GABAergic innervation of interneuron-selective cells in the hippocampus indicates a complex interaction among GABAergic systems, which might ensure the timing and rhythmic synchronization of inhibitory processes in the hippocampus. On the other hand, our results suggest that the serotonergic effect on perisomatic inhibition is exerted via vasoactive intestinal polypeptide-containing basket cells that are functionally distinct from their parvalbumin-positive relatives, which appear to escape control of serotonergic as well as local interneuron-selective cells.     

Immunocytochemical evidence suggests that taurine is colocalized with GABA in the Purkinje cell terminals,but that the stellate cell terminals predominantly contain GABA: a light- and electronmicroscopic study of the rat cerebellum

Summary The distributions of taurine-like and GABA-like immunoreactivities in the rat cerebellum were compared by analysis of consecutive semithin and ultrathin sections, postembedding labeled with the peroxidase-antiperoxidase technique or with an indirect immunogold procedure, respectively. Taurine-like immunoreactivity was selectively enriched in Purkinje cell bodies, dendrites and spines, and boutons in the cerebellar nuclei exhibiting ultrastructural features typical of Purkinje cell terminals. The stellate and basket cell bodies and terminals were very weakly labeled. A computer assisted quantitative assessment of the net immunogold labeling revealed that the mean gold particle density in the Purkinje cell terminals was about 70% higher than that in the Purkinje cell dendrites, and about 14 times higher than that in the stellate/basket cell terminals in the molecular layer. Stellate, basket and Purkinje cell terminals emerged as intensely immunoreactive in adjacent sections processed with an antiserum against conjugated GABA. These findings indicate, contrary to recent electrophysiological data, that GABA is a more likely transmitter candidate than taurine in the stellate cells. The apparent colocalization of GABA and taurine in the terminals of Purkinje cells raises the possibility that these terminals are capable of releasing two different inhibitory amino acids.     

GAD67-GFP+ neurons in the Nucleus of Roller: a possible source of inhibitory input to hypoglossal motoneurons. I. Morphology and firing properties

In this study we examined the electrophysiological and morphological properties of inhibitory neurons located just ventrolateral to the hypoglossal motor (XII) nucleus in the Nucleus of Roller (NR). In vitro experiments were performed on medullary slices derived from postnatal day 5 (P5) to P15 GAD67-GFP knock-in mouse pups. on cell recordings from GFP+ cells in NR in rhythmic slices revealed that these neurons are spontaneously active, although their spiking activity does not exhibit inspiratory phase modulation. Morphologically, GFP+ cells were bi- or multipolar cells with small- to medium-sized cell bodies and small dendritic trees that were often oriented parallel to the border of the XII nucleus. GFP+ cells were classified as either tonic or phasic based on their firing responses to depolarizing step current stimulation in whole cell current clamp. Tonic GFP+ cells fired a regular train of action potentials (APs) throughout the duration of the pulse and often showed rebound spikes after a hyperpolarizing step. In contrast, phasic GFP+ neurons did not fire throughout the depolarizing current step but instead fired fewer than four APs at the onset of the pulse or fired multiple APs, but only after a marked delay. Phasic cells had a significantly smaller input resistance and shorter membrane time constant than tonic GFP+ cells. In addition, phasic GFP+ cells differed from tonic cells in the shape and time course of their spike afterpotentials, the minimum firing frequency at threshold current amplitude, and the slope of their current-frequency relationship. These results suggest that GABAergic neurons in the NR are morphologically and electrophysiologically heterogeneous cells that could provide tonic inhibitory synaptic input to HMs.     

孕期亚硝酸盐暴露对仔鼠海马损伤的影响

目的 探讨孕期亚硝酸盐暴露对海马损伤的作用。 方法 利用C57BL/6J小鼠建立孕期亚硝酸盐暴露模型,分为对照组（生理盐水）、低剂量亚硝酸盐组（60mg/kg）和高剂量亚硝酸盐组(120mg/kg)。收集出生当日（P0）、P7、P14及P30各年龄点仔鼠大脑,用于免疫荧光染色、彗星实验、Western blotting蛋白半定量分析,对海马损伤进行研究。 结果 不论是亚硝酸盐暴露组还是对照组,仔鼠齿状回增殖的神经干细胞数目均随着年龄的增长逐渐减少;在P0、P7、P14和P30年龄点,暴露组仔鼠增殖的神经干细胞的数量明显比对照组少,且具有剂量依赖性（P<0.05,n=96）。为了鉴别这种抑制作用是否具有选择性,我们对P0仔鼠室管膜下区神经干细胞的增殖情况进行了观察。结果发现,亚硝酸盐暴露组室管膜下区的神经干细胞增殖较对照组也减少,同样具有剂量依赖性（P<0.05）;亚硝酸盐暴露组仔鼠门区炎症损伤细胞和凋亡细胞的数目比对照组多,具有剂量依赖性（P<0.05）;彗星实验结果显示,亚硝酸盐暴露组P0仔鼠海马细胞的彗尾比对照组长,具有剂量依赖性(P<0.01);与对照组P0仔鼠相比,亚硝酸盐暴露组仔鼠海马组织内Caspase-8和核因子κB（NF-κB）蛋白的表达量较高（P<0.05）。
结论 孕期亚硝酸盐暴露可通过抑制神经干细胞增殖,促进细胞损伤和凋亡而对仔鼠海马造成损伤。     

Involvement of postnatal apoptosis on sex difference in number of cells generated during late fetal period in the sexually dimorphic nucleus of the preoptic area in rats

Postnatal apoptosis is involved in formation of the sex difference in neuron number of the sexually dimorphic nucleus of the preoptic area (SDN-POA) in rats. In this study, we examined the origin of neurons that die with apoptosis on the postnatal period to exhibit the sex difference in neuron number of the SDN-POA. First, we measured the number of cells that were labeled with 5-bromo-2'-deoxyuridine (BrdU) on embryonic day (ED) 17, ED18, and ED19 in the SDN-POA of rats on postnatal day (PD) 4 and PD8. The SDN-POA had many more cells labeled with BrdU on ED17 and ED18 than those on ED19. Significantly fewer cells labeled with BrdU on ED18 in the female SDN-POA from PD4 to PD8 resulted in a significant sex difference in the number at PD8. Next, combination analyses of BrdU-labeling and immunohistochemistry for single-stranded DNA (ssDNA), an apoptotic marker, were succeeded to investigate whether SDN-POA neurons generated during ED17-18 were removed by apoptosis. Many more ssDNA-immunoreactive cells that had been labeled with BrdU during ED17-18 were found in the SDN-POA of PD8 females, but few in the SDN-POA of PD8 males and PD4 females and males. These results suggest that the sex difference in the number of SDN-POA neurons generated during the late fetal period was caused by postnatal apoptosis.