ENK与CaBPs在PPE-GFP转基因小鼠视网膜细胞中的共存

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)能神经元与钙结合蛋白D28K(CB)、钙视网膜蛋白(CR)和小白蛋白(PV)等钙结合蛋白(CaBPs)成员在视网膜的分布及共存情况。方法:利用免疫组织化学和免疫荧光双标染色的方法。结果:GFP阳性的ENK能细胞主要分布在视网膜内核层内缘,少量分布在节细胞层。所有的GFP阳性细胞均与神经元标志物NSE共存,但不与星形胶质细胞标志物GFAP共存。GFP与CB、CR和PV均有部分共存,其中GFP/CB共存神经元占GFP阳性细胞的8.65%,占CB阳性细胞的5.84%;GFP/CR共存神经元占GFP阳性细胞的18.18%,占CR阳性细胞的14.28%,且共存细胞仅见于内核层;GFP/PV共存细胞占GFP阳性细胞的68.75%,占PV阳性细胞的91.67%,共存细胞主要位于内核层,少量见于节细胞层。结论:ENK能神经元在视网膜内具有板层特异性的分布特点和与钙结合蛋白成员有不同的共存模式,上述结果为深入研究小鼠视网膜ENK能神经元的功能意义提供了形态学依据。     

5-HT1A受体亚型与P物质、Ⅰ型囊泡膜谷氨酸转运体和甘丙肽在大鼠背根神经节神经元中的共表达

为探讨5-HT1A受体亚型参与感觉信息调控的机制,本文利用免疫荧光组织化学双重染色技术观察了该受体亚型与P物质(SP)、I型囊泡膜谷氨酸转运体(VGLUT1)和甘丙肽(Gal)在大鼠背根神经节(DRG)神经元内的共存状况。结果表明:5-HT1A受体亚型阳性神经元占DRG神经元总数的46.2%,阳性神经元以大型及小型神经元为主。在DRG内观察到了5-HT1A/SP、5-HT1A/VGLUT1以及5-HT1A/Gal双标神经元。其中5-HT1A/SP双标神经元占5-HT1A受体亚型阳性神经元的34.6%,占SP阳性神经元的72.0%;5-HT1A/VGLUT1双标神经元占5-HT1A受体亚型阳性神经元的24.1%,占VGLUT1阳性神经元的18.5%;5-HT1A/Gal双标神经元占5-HT1A免疫阳性神经元的17.6%,占Gal免疫阳性神经元的63.8%。5-HT1A/SP和5-HT1A/Gal双标神经元主要为DRG的小型神经元,而5-HT1A/VGLUT1双标神经元主要为大、中型神经元。上述结果提示,5-HT1A受体亚型可能通过调节SP、谷氨酸以及Gal在初级传入终末及外周神经末稍的释放发挥其感觉信息的调节作用。     

大鼠VGLUT2基因shRNA慢病毒载体转染原代培养脊髓神经元对VGLUT2表达的影响

目的:探讨新构建的可以表达针对2型囊泡膜谷氨酸转运体(vesicular glutamate transporter 2,VGLUT2)短发卡RNA(short hairpin RNA,shRNA)的慢病毒载体能否有效地感染大鼠脊髓原代培养神经元,并鉴定其对培养神经元内VGLUT2基因的特异性干扰效果,从而为进一步在整体动物脊髓水平研究VGLUT2的功能提供有力的工具。方法:首先分别将已筛选到的两对互补并靶向作用于编码大鼠VGLUT2序列两个位点的特异性shRNA序列和一个阴性对照的寡核苷酸,克隆到pGCSIL-GFP质粒(经Age I和EcoRI双酶切)载体内,经酶切、测序鉴定及转染293T细胞,包装得到病毒颗粒。然后将筛选出的慢病毒感染体外分离、培养的胚胎大鼠脊髓神经元,将培养的原代神经元分为正常组、阴性对照组、VGLUT2-shRNA-1组和VGLUT2-shRNA-2组,在荧光显微镜下分别观察GFP的表达情况;同时运用Westernt blot检测各组VGLUT2蛋白的表达水平。结果:免疫荧光检测显示,阴性对照组、VGLUT2-shRNA-1组和VGLUT2-shRNA-2组脊髓原代培养神经元均能观察到明显的GFP荧光,表明这些神经元已被慢病毒载体高效转染;但VGLUT2 shRNA-1组和VGLUT2 shRNA-2组神经元VGLUT2的表达量明显低于正常组和阴性对照组。Western blot结果显示,VGLUT2-shRNA-1组和VGLUT2-shRNA-2组的VGLUT2蛋白的表达量与正常组和阴性对照组相比明显有所下降,分别占正常组的62.42±1.12%和66.07±1.33%(P<0.05),但VGLUT2-shRNA-1组和VGLUT2-shRNA-2组之间无显著性差异(P>0.05);阴性对照组与正常组相比亦无明显差异(P>0.05)。结论:VGLUT2-shRNA-1组和VGLUT2-shRNA-2组重组慢病毒载体均能有效地感染原代培养脊髓神经元,并高效下调其神经元内目的基因VGLUT2的表达。     

谷氨酸脱羧酶67-绿色荧光蛋白基因敲入小鼠三叉神经尾侧亚核内GFP阳性神经元的分布及与小白蛋白的共存

目的观察谷氨酸脱羧酶67-绿色荧光蛋白（GAD67-GFP）基因敲入小鼠三叉神经尾侧亚核（Vc）浅层内，表达GFP的GABA能神经元的分布及其与小白蛋白（PV）的共存。方法分别运用原位分子杂交与免疫组织化学相结合；GFP与神经元标记物——神经元核蛋白（NeuN）或PV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进行观察。结果1．Vc浅层内90％以上的GFP阳性神经元同时表达GAD67 mRNA，而几乎所有表达GAD67 mRNA的阳性神经元都呈GFP阳性；2．GFP阳性神经元主要分布于Ｖc的Ⅰ－Ⅱ层内，细胞较小，尤其在Ⅱ层内可见大量密集分布的GFP阳性细胞和突起。GFP阳性神经元分别占Ⅰ、Ⅱ层内NeuN阳性神经元总数的19．4％和24．3％；3．GFP／PV双标神经元主要分布于Ｖc的Ⅰ－Ⅱ层，这些双标神经元大约占PV阳性神经元的62．4％，占GFP阳性神经元的12．8％。结论在Ｖc表达GFP的GABA能神经元主要密集分布于与外周伤害性信息传递关系密切的板层内，且大部分PV样阳性神经元属于GABA能神经元。     

利用GAD67-GFP基因敲入方法显示小鼠脊髓内表达GFP的GABA能神经元的分布(英文)

γ-氨基丁酸 (GABA)是脊髓背角、前角内主要的抑制性神经递质。为了更好地观察脊髓背角内 GABA能神经元的形态和功能 ,本研究使用了两种谷氨酸脱羧酶 67-绿色荧光蛋白 (GAD67-GFP)基因敲入小鼠 ,并观察了敲入小鼠脊髓内的 GFP表达状况。用免疫荧光组织化学双标记方法显示脊髓内所有的 GF P阳性神经元基本上都呈 GAD67和 GABA阳性 ;GFP阳性神经元在脊髓背角的 ～ 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 ,而在脊髓背角其它部位及前角则呈散在分布。脊髓内 GFP阳性神经元的分布与 GABA能神经元的分布一致。本文作者等还进一步在 GAD67-GFP敲入小鼠中观察了 GFP和神经元标志物神经元核蛋白 (Neu N )的共存状况。脊髓背角内 GFP阳性神经元分别占 、 和 层的 Neu N阳性神经元的 3 1.5 %、3 3 .3 %和 44 .7% ,与以往的 GABA免疫组化研究结果基本一致。本研究表明 GAD67-GFP基因敲入小鼠脊髓内的 GFP在GAD67启动子的调节下正确地表达于 GABA能神经元 ,该基因敲入小鼠可用于脊髓 GABA能神经元的形态学特征和生理学特性及其发育规律等方面的研究     

小鼠脊髓背角神经元向臂旁核及丘脑中线/板内核群的分支投射

目的:观察脊髓背角P物质受体(substance P receptor,SPR)阳性神经元向臂旁核及中线/板内核群的分支投射并为其参与痒觉信息传递提供证据。方法:利用小动物脑立体定位仪向10只8周龄C57BL/6小鼠的臂旁核及丘脑中线/板内核群内分别注射四甲基罗达明(tetramethylrhodamine-dextran,TMR)及荧光金(fluoro-gold,FG),观察小鼠脊髓背角的TMR/FG双标神经元。结合免疫荧光组织化学染色方法观察双标神经元表达SPR和在急性痒模型下表达Fos的情况。结果:脊髓背角I层、脊髓外侧核(lateral spinal nucleus,LSN)及脊髓背角IV～V层均可观察到TMR/FG双标神经元,且部分双标神经元分别表达SPR及Fos蛋白。结论:脊髓背角存在同时向臂旁核及丘脑中线/板内核群发出分支投射的神经元,部分双标神经元表达SPR并参与痒觉信息的传递。     

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。     

大鼠脊髓背角内FOS和NDP阳性神经元的分布与联系(英文)

本文应用还原性尼克酰胺腺嘌呤二核苷酸脱氢酶（NDP）组织化学和原癌即刻早期基因c-fos表达产物Fos免疫细 胞化学方法，观察了大鼠脊髓背角内 NDP和 Fos阳性神经元的分布与联系。一侧足跖部皮下注射福尔马林后，同侧背角内可见大量Fos阳性细胞，而对侧背角内未见或偶见Fos阳性细胞。在背角各层中，大多数Fos阳性细胞分布于Ⅰ层及Ⅱ层外带的内侧部．背角内也可见大量NDP阳性胞体，纤维和终末，密集分布于Ⅱ层内带。双标结果说明部分背用内的Fos阳性细胞也是NDP阳性，双标神经元主要分布于区层的内侧部。在Ⅱ层内，Fos阳性细胞周围常有NDP阳性纤维和终末分布，部分NDP阳性终未直接附着干Fos阳性细胞膜上。本文结果为NO参与脊髓内伤害性刺激信息传递过程提供了形态学证据。     

大鼠三叉神经脊束核内Ⅱ型囊泡膜谷氨酸转运体样阳性终末向外侧臂旁核的投射

新近发现的Ⅰ型(VGLUT1)和Ⅱ型(VGLUT2)囊泡膜谷氨酸转运体已被作为谷氨酸能神经终末的标识物,在中枢神经系统呈互补分布,显示二者可能存在功能差异。以往的研究也已经证实接受口面部浅感觉信息的三叉神经脊束核,特别是尾侧亚核(Vc)向外侧臂旁核(LPB)发出丰富的谷氨酸能投射纤维,但该投射纤维内究竟是哪一型的囊泡膜谷氨酸转运体承担谷氨酸的转运功能,到目前为止尚未见报道。本实验选用SD大鼠,综合运用顺行束路追踪[将霍乱毒素B亚单位-CTb分别注入一侧Vc、三叉神经脊束核极间亚核(Vi)、吻侧亚核(Vo)]和免疫荧光组织化学相结合的三重标记技术,在激光共聚焦显微镜下对Vc、Vi和Vo内向LPB投射纤维内所含的囊泡膜谷氨酸转运体(VGLUTs)的情况进行了研究。结果显示:(1)将CTb分别注入大鼠一侧Vc、Vi或Vo后,在同侧的LPB内可观察到许多CTb顺行标记纤维,其中以来自Vc的标记纤维最多,Vi次之,Vo最少;(2)在LPB内可见部分CTb顺标终末同时表达VGLUT2样免疫阳性,但未见与VGLUT1样阳性终末的共存;(3)可观察到部分CTb/VGLUT2双标终末散在分布于NeuN标记的神经元的胞体周围,并与之形成密切接触。以上结果提示:大鼠口面部浅感觉、特别是伤害性信息从三叉神经脊束核向LPB传递的过程中,谷氨酸发挥着重要作用,负责其内谷氨酸转运功能的主要是VGLUT2。     

大鼠延髓背角内5-羟色胺、脑啡肽、γ-氨基丁酸、甘氨酸或P-物质能终末与钙结合蛋白阳性神经元间的联系(英文)

CB(calbindin-D28k),CR(calretinin)和PV(parvalbumin)是最常见的3种钙结合蛋白(calcium-binding proteins,CaBPs)。本研究首先观察了面口部给予伤害性刺激诱发大鼠延髓背角(又称三叉神经脊束核尾侧亚核)神经元表达FOS蛋白的状况;然后通过免疫荧光组织化学技术检测这些神经元内是否含有CaBPs(CB、CR和PV);最后通过免疫荧光和免疫电镜染色技术观察5-HT、GABA、甘氨酸转运体2(glycine transporter 2,GlyT2)、脑啡肽(enkephalin,ENK)或SP与CaBPs/FOS双标神经元间的联系。在光镜下可观察到:(1) FOS阳性神经元在延髓背角各层均有分布,以Ⅱ层最为密集;(2)大多数CB、CR或PV阳性神经元位于Ⅱ层,余者分布在Ⅰ层和Ⅲ层;(3) 5-HT、GABA、GlyT2,ENK及SP阳性纤维和终末主要位于延髓背角浅层(4)部分FOS阳性神经元同时呈CB、CR或PV阳性;(5) 5-HT、GABA、GlyT2或ENK阳性终末分别与FOS/CB、FOS/CR或FOS/PV双标神经元形成密切接触;(6) SP阳性终末与5-HT、GABA、GlyT2或ENK阳性终末同时与CB、CR或PV阳性神经元形成密切接触。在电镜下观察到5-HT、GABA、GlyT2或ENK阳性终末与CB、CR或PV阳性神经元主要形成对称型(抑制性)突触联系。这些结果提示在大鼠延髓背角,5-HT、GABA、甘氨酸或ENK可能通过抑制含钙结合蛋白的伤害性感受神经元来调节面口部伤害性信息的传递。     

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.     

Preproenkephalin mRNA is Expressed in a Subpopulation of GABAergic Neurons in the Spinal Dorsal Horn of the GAD67‐GFP Knock‐In Mouse

GABA (γ‐aminobutyric acid)ergic neurons in the spinal dorsal horn have been reported to be divided into distinctive populations, with different cotransmitters and neuropeptides. In this study, we examined the colocalization of enkephalin (ENK) mRNA with GABA in the spinal dorsal horn using the glutamic acid decarboxylase (GAD)₆₇‐green fluorescence protein (GFP) knock‐in mouse. Our approach was to perform in situ hybridization histochemistry to detect mRNA for preproenkephalin (PPE, the precursor protein for ENK), combined with immunohistochemistry for GFP to reveal GABAergic neurons. Quantitative analysis indicated that more than 44.4% (2967/6681) of GFP‐immunoreactive neurons showed signals for PPE mRNA in the spinal dorsal horn. While 53.9% (2967/5501) of PPE mRNA‐expressing neurons were immunoreactive for GFP. The double‐labeled neurons were observed throughout the spinal dorsal horn, although they had a preferential localization in superficial layers. The present results provide a detailed morphological evidence that ENK and GABA colocalized in a subpopulation of neurons in the spinal dorsal horn, which are likely to represent local inhibitory dorsal horn interneurons involved in the modulation of pain transmission. Anat Rec, 291:1334–1341, 2008. © 2008 Wiley‐Liss, Inc.     

大鼠脊髓内囊泡膜谷氨酸转运体1和囊泡膜谷氨酸转运体2阳性纤维和终末在生后发育中的分布和表达变化

目的 探讨囊泡膜谷氨酸转运体1（VGLUT1）和VGLUT2阳性纤维和终末在生后第0天（P0）至第22天（P22）大鼠脊髓内的分布情况和表达变化。 方法 对生后发育P0～P22大鼠的颈膨大和腰膨大部位,进行VGLUT1和VGLUT2免疫组织化学染色。 结果 P0～P22大鼠颈膨大和腰膨大脊髓内均可观察到VGLUT1和VGLUT2阳性纤维和终末,但未观察到胞体样结构。VGLUT1和VGLUT2阳性纤维和终末的分布呈现明显的互补分布,尤其是以脊髓后角更加明显。其中,VGLUT1阳性纤维和终末在P0主要见于颈膨大和腰膨大脊髓后角Ⅲ～Ⅴ层,中间部和前角很微弱。脊髓发育至P3,不仅Ⅲ~Ⅴ层VGLUT1的表达进一步增强,且向外侧部扩展,并在后角基底部Ⅵ层和前角的外侧部（Ⅸ层）也可观察到较强的VGLUT1阳性纤维,呈现一条明显由背内向腹外的带状分布趋势。P7时此带状分布更加明显,并随着发育逐渐向内、外扩展,至P22时已广泛分布于除Ⅱ层之外的整个脊髓。而VGLUT2阳性纤维和终末在P0时即密集出现于脊髓后角Ⅰ～Ⅱ层以及前角的外侧边缘区域;之后随着发育,VGLUT2阳性纤维和终末的分布模式并未发生明显改变,但其密度逐渐有所增加,特别是Ⅰ～Ⅱ层内VGLUT2阳性产物的表达尤为明显。另外,在脊髓白质后索内可见VGLUT1阳性皮质脊髓后束纤维由颈髓（P3）逐渐下降至腰髓（P7）的发育过程。 结论 VGLUT1和VGLUT2阳性纤维和终末在脊髓发育过程中呈现明显不同,且表现出互补分布的特点,这对于进一步理解VGLUT1和VGLUT2在脊髓生后发育过程中不同功能特点可能有意义。     

Propriospinal neurons with ascending collaterals to the dorsal medulla,the thalamus and the tectum: a retrograde fluorescent double-labeling study of the cervical cord of the rat

Summary Branching neurons with descending propriospinal collaterals and ascending collaterals to the dorsal medulla, the thalamus and the tectum were studied in the rat's cervical spinal cord (C1–C8), using the retrograde fluorescent double-labeling technique: Diamidino Yellow Dihydrochloride (DY) was injected in the cord at T2, True Blue (TB) was injected in the brain stem. DY-labeled descending propriospinal neurons were present in all laminae, except lamina IX. They were concentrated in lamina I, laminae IV to VIII, and in the lateral spinal nucleus, LSN. TB-labeled neurons projecting to the dorsal medulla were concentrated in lamina IV and the medial parts of laminae V and VI (probably representing postsynaptic dorsal column — PSDC — neurons), but were also present in lamina I, the LSN, the lateral dorsal horn, and in laminae VII and VIII. DY-TB double-labeled neurons giving rise to both a descending propriospinal collateral and an ascending collateral to the dorsal medulla were intermingled with the TB single-labeled neurons. About 4% of the descending propriospinal neurons gave rise to an ascending collateral to the dorsal column nuclei; these double-labeled cells constitute a sizable fraction (10%) of the PSDC neurons. TB-labeled spinothalamic and spinotectal neurons were located in lamina I, the lateral cervical nucleus (LCN), the LSN, the lateral lamina V, lamina VII and VIII, lamina X and in the spinal extensions of the dorsal column nuclei, predominantly contralateral to the TB injections. DY-TB double-labeled neurons were present throughout C1–C8 in the LSN, lateral lamina V, lamina VIII, ventromedial lamina VII, and lamina X. Only very few were observed in lamina I and the LCN, and none in the spinal extensions of the dorsal column nuclei. The double-labeled neurons constituted only a minor fraction of all labeled neurons; 3–5% of the spinothalamic neurons and about 1–7% of the spinotectal neurons were double-labeled. Conversely, only about 1% of the labeled descending propriospinal neurons gave rise to an ascending spinothalamic collateral, and even fewer (0.1 to 0.6%) to a collateral to the dorsal midbrain. The LSN displayed the highest relative content of branching neurons. Up to 20% of its ascending spinothalamic and spinotectal neurons and up to 8% of its descending propriospinal neurons were found to be branching neurons, indicating that the LSN constitutes an unique cell-group in the rat spinal cord.     

The neuropeptide tyrosine Y1R is expressed in interneurons and projection neurons in the dorsal horn and area X of the rat spinal cord

The localization of the neuropeptide tyrosine Y1 receptor was studied with immunohistochemistry in parasagittal and transverse, free-floating sections of the rat lumbar spinal cord. At least seven distinct Y1 receptor-positive populations could tentatively be recognized: Type 1) abundant small, fusiform Y1 receptor-positive neurons in laminae I-II, producing a profuse neuropil; Type 2) Y1 receptor-positive projection neurons in lamina I; Type 3) small Y1 receptor-positive neurons in lamina III, similar to Type 1 neurons, but less densely packed; Type 4) a number of large, multipolar Y1 receptor-positive neurons in the border area between laminae III-IV, with dendrites projecting toward laminae I-II; Type 5) a considerable number of large, multipolar Y1 receptor-positive neurons in laminae V-VI; Type 6) many large Y1 receptor-positive neurons around the central canal (area X); and Type 7) a small number of large Y1 receptor-positive neurons in the medial aspect of the ventral horns (lamina VIII). Many of the neurons present in laminae V-VI and area X produce craniocaudal processes extending for several hundred micrometers. Retrograde tracing using cholera toxin B subunit injected at the 9th thoracic spinal cord level shows that several Type 5 neurons in laminae V-VI, and at least a few Type 2 in lamina I and Type 6 in area X have projections extending to the lower segments of the thoracic spinal cord (and perhaps to supraspinal levels). The present results define distinct subpopulations of neuropeptide tyrosine-sensitive neurons, localized in superficial and deep layers of the dorsal, in the ventral horns and in area X. The lamina II neurons express somatostatin [The neuropeptide Y Y1 receptor is a somatic receptor on dorsal root ganglion neurons and a postsynaptic receptor on somatostatin dorsal horn neurons. Eur J Neurosci 11:2211-2225] and are presumably glutamatergic [Todd AJ, Hughes DI, Polgar E, Nagy GG, Mackie M, Ottersen OP, Maxwell DJ (2003) The expression of vesicular glutamate transporters VGLUT1 and VGLUT2 in neurochemically defined axonal populations in the rat spinal cord with emphasis on the dorsal horn. Eur J Neurosci 17:13-27], that is they are excitatory interneurons under a Y1 receptor-mediated inhibitory influence. The remaining Y1 receptor-positive spinal neurons need to be phenotyped, for example if the large Y1 receptor-positive laminae III-IV neurons (Type 5) are identical to the neurokinin (NK)1R-positive neurons previously shown to receive neuropeptide tyrosine positive dendritic contacts [Polgár E, Shehab SA, Watt C, Todd AJ (1999) GABAergic neurons that contain neuropeptide Y selectively target cells with the NK1 receptor in laminae III and IV of the rat spinal cord. J Neurosci 19:2637-2646]. If so, neuropeptide tyrosine could have an antinociceptive action not only via Y1 receptor-positive interneurons (Type 1) but also projection neurons. The present results show neuropeptide tyrosine-sensitive neuron populations virtually in all parts of the lumbar spinal cord, suggesting a role for neuropeptide tyrosine signaling in many spinal functions, including pain.