神经元型一氧化氮合酶的神经生物学研究进展

神经元型一氧化氮合酶(neuronal nitric oxide synthase,nNOS)主要表达于神经元,在星形胶质细胞和神经干细胞中也有一定水平的表达.不同的mRNA拼接形式产生了nNOS蛋白的5种亚型,包括nNOS-α、nNOS-β、nNOS-μ、nNOS-γ和nNOS-2.nNOS单体不具催化活性,二聚体是...     

Preso调控nNOS在神经元机械性损伤中的作用

目的通过建立神经元机械性损伤模型,研究突触后致密物质(PSD)支架蛋白中的树突棘突触后致密物质-95(PSD-95)相互作用调节蛋白(Preso)在创伤性脑损伤中的作用及调控机制。方法建立神经元机械性损伤模型,通过细胞活力和乳酸脱氢酶(LDH)检测神经元损伤程度,并通过Western blot检测明确Preso在损伤后的表达变化;利用Preso慢病毒过表达载体上调Preso在神经元中的表达,通过细胞活力和LDH检测明确上调Preso在神经元损伤中的作用;利用神经元特异性一氧化氮合酶(nNOS)特异性抑制剂ARL 17477,通过细胞活力和LDH检测明确抑制nNOS对上调Preso调控神经元损伤的影响。结果神经元机械性损伤后,Preso表达无明显改变,而上调Preso表达可加重神经元损伤;利用nNOS特异性抑制剂ARL 17477可显著改善神经元损伤,并抑制上调Preso表达对神经元损伤的影响。结论神经元机械性损伤后,Preso可促进神经元损伤的形成,而nNOS是其重要的下游效应分子。     

颞叶癫痫大鼠突触后致密物中差异性表达蛋白的筛选和鉴定

目的 筛选和鉴定颞叶癫痫(temporal lobe epilepsy,TLE)大鼠模型突触后致密物(postsynaptic density,PSD)中差异性表达的蛋白质.方法 建立氯化锂-毛果芸香碱TLE大鼠模型,收集TLE组和健康对照组大鼠新鲜全脑组织,联合应用蔗糖密度梯度离心和膜顺序提取技术提取PSD蛋白质,进行双向凝胶电泳,通过PDQuest图像分析软件筛选TLE组差异性表达的蛋白质点,并应用基质辅助激光解析电离飞行时间质谱(MALDI-TOF-MS)对其进行鉴定.结果 与健康对照组比较,TLE组存在70个差异性表达PSD蛋白质点,其中表达上调的蛋白质主要包括果糖二磷酸醛缩酶、肌酸激酶和热休克蛋白27,表达下调的蛋白质主要包括微管蛋白、肌动蛋白、中间丝蛋白α和脯氨酰顺反异构酶.结论 TLE大鼠PSD中存在差异性表达蛋白质,其异常表达可能与TLE的形成、发作或神经元保护有关.     

血小板活化因子通过N-甲基-D-天(门)冬氨酸/突触后密度蛋白93途径致神经元损伤

目的探讨血小板活化因子(PAF)所致的神经元损伤是否涉及N-甲基-D-天(门)冬氨酸／突触后密度蛋白93(NMDA／PSD93)信号通路。方法细胞培养系统培养原代野生型和PSD93基因敲除型小鼠皮质神经元;0．3umol／LPAF处理神经元24h或5umol／LPAF受体拮抗剂(BN52021),10umol／L非竞争性NMDA受体拈抗剂(MK-801)和60umol／L神经性一氧化氮合成酶(nNOS)抑制剂(L—NAMA)预处理,碘化物／钙黄绿素染色检测细胞凋亡;免疫印迹检测野生型和基因敲除型小鼠皮质神经元中的多种蛋白表达;细胞免疫组化和共聚焦显微镜观察在同一神经轴突上共同表达多种蛋白;放免法测定神经元细胞蛋白中环鸟苷磷酸(cGMP)活性。结果(1)PSD93基因敲除型神经元不表达PSD93外,与野生型一样表达PSD93N-甲基-D-天(门)冬氨酸受体(NR2A)和nNOS。(2)神经轴突共同表达PSD93、NR2A和nNOS。(3)PSD93基因敲除型小鼠皮质神经元减少PAF对神经的毒性作用,并降低其cGMP活性。结论PAF通过NMDA／PSD93途径致神经细胞损伤。     

突触后密度蛋白93基因敲除抑制血小板活化因子性神经元损害

目的　探讨血小板活化因子 (PAF)所致的神经元毒性损伤是否受突触后密度蛋白 93(PSD93 )基因调控。方法　用PAF处理培养的野生型和PSD93 基因敲除型的小鼠皮质神经元 ;经碘化物 (PI) /钙黄绿素(Calcein)染色 ,荧光显微镜观察并计算细胞死亡率 (% ) ;细胞生存能力检测 (MTT)神经元生存能力 ;免疫印迹法测定PSD93 和PSD95蛋白的表达。结果　野生型神经元表达PSD93 和PSD95蛋白 ,PSD93 基因敲除型只表达PSD95,不表达PSD93 ;基因敲除型的小鼠皮质神经元凋亡明显低于野生型 (P <0 0 5 ) ,生存能力高于野生型(P <0 0 1)。结论　PSD93 基因敲除抑制PAF所致的神经元损害     

小檗碱对三转基因阿尔茨海默病小鼠的学习记忆和海马PSD95突触相关蛋白表达水平的影响

目的 探讨小檗碱(BBR)对三转基因阿尔茨海默病(AD)小鼠的学习记忆及海马组织PSD95突触蛋白表达水平的影响。方法 将30只三转基因(APP/Tau/PS1)AD小鼠按随机数字表法分成3组,即AD对照组、AD+25 mgBBR组、AD+50 mgBBR,每组各10只,后2组以灌胃方式且剂量分别为25 mg·kg^-1·d^-1、50 mg·kg^-1·d^-1,对照组给予等剂量生理盐水连续3个月灌胃处理; 采用Morris水迷宫方法探测各组AD小鼠行为学改变、空间记忆及探索情况; 免疫荧光染色检测各组小鼠海马组织突触后致密蛋白95(PSD95)阳性表达水平; Western blotting(WB)法检测各组三转基因AD小鼠海马脑组织PSD95蛋白、磷酸化蛋白激酶B(p-Akt)和磷酸化雷帕霉素靶蛋白(p-mTOR)表达水平及微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)自噬水平。结果 AD+25 mgBBR组的逃避潜伏期的学习记忆能力、免疫荧光PSD95表达水平以及PSD995、LC3-Ⅱ、p-Akt、p-mTOR蛋白表达水平与AD对照组比较均有明显差异(P<0.05); AD+50 mgBBR组逃避潜伏期的学习记忆能力、免疫荧光PSD95表达水平以及LC3-Ⅱ、p-Akt、p-mTOR表达水平与AD对照组比较差异均更明显(P<0.05,P<0.01)。结论 应用50 mg小檗碱能较好改善三转基因AD小鼠的学习记忆、空间探索能力,其机制可能是通过增加自噬水平LC3-Ⅱ调控Akt/mTOR信号通路,增加突触蛋白PSD95的表达水平及突触数量,以改善AD相关临床症状。     

ATP在Aβ_(1-42)导致的突触可塑性损害中的作用

目的探讨ATP在调节神经元突触可塑性中的作用。方法培养大鼠原代神经元细胞,应用Aβ_(1-42)孵育原代培养的神经元细胞48 h,或者在Aβ_(1-42)孵育原代培养的神经元细胞前30 min预先给予ATP处理细胞。应用Alexa Fluor 488-phalloidin dye染色观察不同处理后神经元树突棘的变化。同时,应用Western blot检测Aβ_(1-42)及ATP处理后对PDS-95蛋白表达的影响。结果 ATP能减少Aβ_(1-42)所导致的神经元树突棘丢失,Aβ_(1-42)孵育原代培养的神经元细胞48 h后,PSD-95蛋白水平降低;而经过ATP预处理30 min后,神经元PSD-95蛋白表达无显著变化。结论 ATP能够调节神经元突触可塑性,从而发挥脑保护作用。     

星形胶质细胞对天冬氨酸特异性半胱氨酸蛋白酶介导β淀粉样蛋白早期突触毒性作用的影响

目的探讨星形胶质细胞(astrocyte,AS)对天冬氨酸特异性半胱氨酸蛋白酶(cysteinyl aspartate specific proteinase,caspase)介导β淀粉样蛋白(β-amyloid,Aβ)早期突触毒性作用的影响,以期为进一步研究与血管性痴呆(vascular dementia,Va D)的发病机制奠定基础。方法以原代培养大鼠海马纯神经元体系(NE-S)及混合培养体系(MIX-S,主要包含神经元及AS)为研究对象,各体系分为6组:对照组、caspase-8抑制剂组、caspase-9抑制剂组、Aβ处理组、caspase-8抑制剂预处理加Aβ组和caspase-9抑制剂预处理加Aβ组。免疫荧光检测各组近胞体10μm段树突中突触后密度蛋白(postsynaptic density-95,PSD95)表达量的变化。结果 1在NE-S与MIX-S中,与对照组相比,caspase-8抑制剂组、caspase-9抑制剂组PSD95的表达量均无明显差异,Aβ处理组PSD95的表达量均显著降低(P均0.001)。2在NE-S中,与Aβ处理组相比,caspase-9抑制剂预处理加Aβ组PSD95的表达量显著回升至对照组水平,caspase-8抑制剂预处理加Aβ组则无显著改变;在MIX-S中的结果则相反,即caspase-8抑制剂预处理加Aβ组PSD95的表达量显著回升至对照组水平,而caspase-9抑制剂预处理加Aβ组则无显著改变。3MIX-S与NE-S两种培养系统间相比较,对照组间及Aβ处理组间PSD95的表达量均无显著差异,而caspase-8抑制剂预处理加Aβ组间及caspase-9抑制剂预处理加Aβ组间PSD95的表达量差异有显著性。结论在Aβ早期突触毒性作用中,AS参与caspase-8介导的死亡受体通路激活过程,且参与抑制神经元的线粒体通路。     

热休克蛋白70的功能及其临床应用前景

正热休克蛋白(hot shock proteins,Hsp)为转录调节蛋白热休克因子(hot shock factor,HSF)诱导其相关基因启动子上存在的热休克原件(hot shockelement,HSE)基因而产生的一类蛋白。脑缺血能引发Hsp反应。Hsp70在正常脑组织中表达水平极低,在缺血脑组织神经元中却被诱导表达,尤其在缺血病灶周边,通过与ATP结合能够对错误折叠和未折     

突触后支架蛋白与神经退行性疾病

突触后致密物质作为神经元兴奋性突触后膜上的特殊结构,在神经元功能调节中具有重要作用。PSD-95、Shank、Homer是突触后致密物质中重要的支架蛋白,参与调节神经元信号传导、突触可塑性等过程,与神经系统疾病的发生和发展密切相关。该文就突触后支架蛋白在阿尔兹海默症、帕金森病等神经退行性疾病中的作用及机制进行综述,以此探讨突触后支架蛋白及其相关信号通路作为神经退行性疾病靶点治疗的可能性。     

The protective effect of neuronal nitric oxide synthase (nNOS) against alcohol toxicity depends upon the NO-cGMP-PKG pathway and NF-kappaB

Fetal alcohol syndrome (FAS) stems from maternal alcohol abuse during pregnancy and is an important cause of mental retardation and hyperactivity in children. In the developing brain, alcohol can kill neurons, leading to microencephaly. However, due to their genetic makeup, some individuals are less vulnerable than others to alcohol's neurotoxic effects. Animal studies have demonstrated that one particular gene, neuronal nitric oxide synthase (nNOS), protects developing neurons in vivo against alcohol-induced death. We utilized pharmacologic techniques to demonstrate that nNOS protects neurons against alcohol toxicity by activating the NO-cGMP-PKG signaling pathway. Cerebellar granule cell cultures derived from mice carrying a null mutation for nNOS (nNOS-/- mice) were substantially more vulnerable than cultures from wild-type mice to alcohol-induced cell death. However, activation of the pathway at sites downstream of nNOS protected the cultures against alcohol toxicity. Conversely, blockade of the pathway rendered wild-type cultures vulnerable to alcohol-induced death. We further identified NF-kappaB as the downstream effector through which nNOS and the NO-cGMP-PKG pathway signal their neuroprotective effects. Tumor necrosis factor-alpha (TNF-alpha), which activates NF-kappaB, ameliorated alcohol-induced cell death in nNOS-/- and wild-type cultures, while an NF-kappaB inhibitor (NFi) blocked the protective effects of TNF-alpha and worsened alcohol-induced cell death. Furthermore, NFi blocked the protective effects of NO-cGMP-PKG pathway activators, demonstrating that NF-kappaB is downstream of the NO-cGMP-PKG pathway. As wild-type neurons matured in culture, they became resistant to alcohol toxicity. However, this maturation-dependent alcohol resistance did not occur in nNOS-/- mice and could be reversed in wild-type mice with NFi, demonstrating that nitric oxide and NF-kappaB are crucial for the development of alcohol resistance with age. Thus, nNOS protects developing neurons against alcohol toxicity by activating the NO-cGMP-PKG-NF-kappaB pathway and is crucial for the acquisition of maturation-dependent alcohol resistance.     

Severe alcohol-induced neuronal deficits in the hippocampus and neocortex of neonatal mice genetically deficient for neuronal nitric oxide synthase (nNOS)

Alcohol can severely damage the developing brain, and neuronal loss is a critical component of this injury. Thus, identification of molecular factors that ameliorate alcohol-induced neuronal loss is of great importance. Previous in vitro work has demonstrated that nitric oxide (NO) protects neurons against alcohol toxicity. We tested the hypothesis that neonatal mice carrying a null mutation for neuronal nitric oxide synthase (nNOS), the enzyme that synthesizes NO in neurons, have an increased vulnerability to alcohol-induced neuronal loss in the neocortex and hippocampus. Wildtype mice and nNOS-/- mice received ethanol (0.0, 2.2, 3.3, or 4.4 g/kg) daily over postnatal days (P) 4-9 and were sacrificed on P10. The number of hippocampal CA1 and CA3 pyramidal cells, dentate gyrus granule cells, and neocortical neurons were determined using stereological methods. Alcohol pharmacokinetics did not differ between wildtype and nNOS-/- strains. Alcohol induced dose-dependent reductions in all four neuronal populations, and the losses were substantially more severe in the nNOS-/- mice than in wildtype. Furthermore, the threshold dose of alcohol to induce cell death was lower in the nNOS-/- mice than in the wildtype mice for all neuronal populations. While nNOS deficiency worsened alcohol-induced neuronal losses, the magnitude of this exacerbation varied among brain regions and depended on alcohol dose. These results demonstrate that nNOS deficiency decreases the ability of developing neurons in vivo to survive the toxic effects of alcohol and strengthen the hypothesis that NO exerts a neuroprotective effect against alcohol toxicity in the developing brain.     

Contributions of endothelial and neuronal nitric oxide synthases to cerebrovascular responses to hyperoxia.

Hyperoxia causes a transient decrease in CBF, followed by a later rise. The mediators of these effects are not known. We used mice lacking endothelial or neuronal nitric oxide synthase (NOS) isoforms (eNOS-/- and nNOS-/- mice) to study the roles of the NOS isoforms in mediating changes in cerebral vascular tone in response to hyperoxia. Resting regional cerebral blood flow (rCBF) did not differ between wild type (WT), eNOS-/- mice, and nNOS-/- mice. eNOS-/- mice showed decreased cerebrovascular reactivities to NG-nitro-L-arginine methyl ester (L-NAME), PAPA NONOate, acetylcholine (Ach), and SOD1. In response to hyperbaric oxygen (HBO2) at 5 ATA, WT and nNOS-/- mice showed decreases in rCBF over 30 minutes, but eNOS-/- mice did not. After 60 minutes HBO2, rCBF increased more in WT mice than in eNOS-/- or nNOS-/- mice. Brain NO-metabolites (NOx) decreased in WT and eNOS-/- mice within 30 minutes of HBO2, but after 45 minutes, NOx rose above control levels, whereas they did not change in nNOS-/- mice. Brain 3NT increased during HBO2 in WT and eNOS-/- but did not change in nNOS-/- mice. These results suggest that modulation of eNOS-derived NO by HBO2 is responsible for the early vasoconstriction responses, whereas late HBO2-induced vasodilation depends upon both eNOS and nNOS.     

Ischemic nitric oxide and poly (ADP-ribose) polymerase-1 in cerebral ischemia: male toxicity, female protection.

It is well established that tissue damage and functional outcome after experimental or clinical stroke are shaped by biologic sex. We investigated the novel hypothesis that ischemic cell death from neuronally derived nitric oxide (NO) or poly-ADP ribose polymerase (PARP-1) activation is sexually dimorphic and that interruption of these molecular death pathways benefits only the male brain. Female neuronal nitric oxide synthase (nNOS) knockout (nNOS-/-) mice exhibited exacerbated histological injury after middle cerebral artery occlusion (MCAO) relative to wild-type (WT) females, unlike the protection observed in male nNOS-/- littermates. Similarly, treatment with the nNOS inhibitor (7-nitroindozole, 25 mg/kg) increased infarction in female C57Bl6 WT mice, but protected male mice. The mechanism for this sexually specific response is not mediated through changes in protein expression of endothelial NOS or inducible NOS, or differences in intraischemic cerebral blood flow. Unlike male PARP-1 knockouts (PARP1-/-), female PARP1-/- littermates sustained grossly increased ischemic damage relative to sex-matched WT mice. Treatment with a PARP inhibitor (PJ-34, 10 mg/kg) resulted in identical results. Loss of PARP-1 resulted in reversal of the neuroprotective activity by the female sex steroid, 17beta estradiol. These data suggest that the previously described cell death pathways involving NO and PARP ischemic neurotoxicity may be operant solely in male brain and that the integrity of nNO/PARP-1 signaling is paradoxically protective in the female.     

Nitric oxide synthase in the hypothalamic paraventricular nucleus of the female rat; organization of spinal projections and coexistence with oxytocin or vasopressin

We investigated the distributions and interrelations of neuronal nitric oxide (NO) synthase- (nNOS), oxytocin- (OT), and 8-arginine vasopressin- (AVP) immunoreactive (IR) neurons in the paraventricular nucleus (PVN), and the occurrence and distribution of nNOS spinally projecting neurons in the PVN of the female rat. Using double labelling immunohistochemistry, we mapped the distribution of nNOS-, OT- and AVP-immunoreactive (IR) neuronal cell bodies in the different parts of the PVN. About 80% of nNOS-IR cell bodies were magnocellular. About 30% of the nNOS-IR cell bodies were OT-IR, colocalization being most frequent in the rostral parts. In comparison, only approximately 3% of all nNOS-IR cell bodies were AVP-IR, evenly distributed throughout the PVN. True Blue (TB), administered unilaterally into the spinal cord, disclosed that most spinally projecting cell bodies in the PVN were localized in caudal parts. Combined TB tracing and nNOS immunohistochemistry showed that approximately 30% of spinally projecting neurons in the PVN were nNOS-IR, and that approximately 40% of these were magnocellular. Ipsilateral nNOS spinal projections were about eight times more frequent than the contralateral nNOS projections. The study describes the detailed neuroanatomical organization of nNOS neurons coexpressing OT or AVP, and of nNOS spinally projecting neurons within defined parts of the PVN. In contrast to the paraventriculo-spinal system in general, we show that the nNOS paraventriculo-spinal pathway to a large extent originates in magnocellular cell bodies. The results suggest that NO is an important messenger in the paraventriculo-spinal pathway that may in part act in concert with OT.     

Role of nitric oxide in cerebral blood flow changes during kainate seizures in mice: genetic and pharmacological approaches

The role of neuronal nitric oxide (NO) in the cerebrovascular response to partial seizures induced by intrahippocampal injection of kainate (KA) was investigated in mice deleted for the neuronal NO synthase gene (nNOS-/-) and in wild-type controls (WT). A second group of WT mice received the nNOS inhibitor, 7-nitroindazole (WT-7NI). Local cerebral blood flow (LCBF) was measured using the quantitative (14)C-iodoantipyrine method. Within the epileptic focus, all three groups of seizing mice (WT, WT-7NI, and nNOS-/-) showed significant 26-88% LCBF increases in ipsilateral hippocampus, compared to saline-injected mice. Contralaterally to the epileptic focus, KA seizures induced a 21-47% LCBF decreases in hippocampus and limbic cortex of WT mice and in most contralateral brain structures of nNOS-/- mice, while WT-7NI mice showed no contralateral CBF change. Neuronal NO appears to be not involved in the cerebrovascular response within the epileptic focus, but may rather have a role in the maintenance of distant LCBF regulation during seizures.     

Anesthetic concentrations of riluzole inhibit neuronal nitric oxide synthase activity, but not expression, in the rat hippocampus

We hypothesized that anesthetic dose of riluzole, an inhibitor of glutamate neurotransmission, may affect the activity and/or expression of neuronal NOS (nNOS). Riluzole, N(G)-nitro-L-arginine-methyl ester (L-NAME) and 7-nitro indazole (7-NI) produced a concentration-related inhibition of nNOS activity in vitro. Riluzole competed with 7-NI for inhibition of nNOS activity, but had no effect on nNOS or endothelial NOS (eNOS) protein expression. Also, nNOS activity was significantly decreased in riluzole-anesthetized rats (40 mg kg(-1) i.p., -32+/-6% from controls, P<0.05). Therefore, blockade of nNOS activity may be involved in the anesthetic effects of riluzole in vivo.     

Stroke outcomes in mice lacking the genes for neuronal heme oxygenase-2 and nitric oxide synthase

Heme oxygenase-2 (HO-2) has been suggested to be a cytoprotective enzyme in a variety of in vivo experimental models. HO-2, the constitutive isozyme, is enriched in neurons and, under normal conditions, accounts for nearly all of brain HO activity. HO-2 deletion (HO-2-/-) leads to increased neurotoxicity in cultured brain cells and increased damage following transient cerebral ischemia in mice. Moreover, pharmacologic inhibition of HO activity significantly augments focal ischemic damage in wildtype (WT) mice, but does not further exacerbate it in HO-2-/- mice. The HO system shares some similarities with nitric oxide synthase (NOS), notably their syntheses of carbon monoxide (CO) and nitric oxide (NO), respectively, which are diffusible gases with numerous biological actions, including neurotransmission and vasodilation. While deletion of HO-2 results in greater stroke damage, the pharmacologic inhibition of neuronal nitric oxide synthase (nNOS), or its gene deletion, confers neuroprotection in animal models of transient cerebral ischemia. To investigate the interactions, the outcome of focal cerebral ischemia-reperfusion in double knockout (HO-2-/- X nNOS-/-) mice lacking both genes was compared to control WT mice. Wildtype and double knockout male mice underwent intraluminal middle cerebral occlusion for 2 hours, followed by reperfusion for 22 hours. Outcomes in neurologic deficits and infarct size were determined. No difference was observed between WT and double knockout mice in the volume of infarction, neurologic signs, decrease in relative cerebral blood flow during ischemia, or core body temperature. The results suggest that the deleterious action of nNOS would counteract the role of HO-2 in neuroprotection.     

Pleiotropic contributions of nitric oxide to aggressive behavior

Male mice with targeted deletion of the genes encoding the neuronal (NOS-1-/- or nNOS-/-) isoform of nitric oxide synthase display altered aggressive behaviors. Male nNOS-1-/- mice are more aggressive than wild-type (WT) mice in all testing paradigms. Testosterone is necessary, but not sufficient, for evoking the persistent aggression, and that serotonin (5-HT) metabolism is altered in male nNOS-1-/- mice. The specific deletion of the nNOS-1 gene not only results in a lack of nNOS-1 protein, but in common with many genes, affects several 'down-stream' processes. In this review, we address whether the elevated aggression in male nNOS-1-/- mice reflects pleiotropic effects of the nNOS-1 gene on pain sensitivity, 'anxiety-like', or 'depressive-like' behaviors. For example, male nNOS-1-/- mice display increased sensitivity to painful stimuli, which may prolong aggressive interactions. Despite elevated corticosterone concentrations, nNOS-1 knockout mice appear to be less 'anxious' or fearful than WT mice. Male nNOS-1-/- mice display longer latencies to right themselves on an inverted platform and spend more time in the center of an open field than WT mice. Because of reduced serotonin turnover, the excessive aggressiveness displayed by nNOS-1-/- mice may be symptomatic of a depressive-like syndrome. However, nNOS-1-/- mice rarely display behavioral 'despair' when assessed with the Porsolt forced swim test; rather, nNOS-1-/- mice show vigorous swimming throughout the assessment suggesting that the aggressive behavior does not represent depressive-like behavior. Importantly, aggressive behavior is not a unitary process, but is the result of complex interactions among several physiological, motivational, and behavioral systems, with contributions from the social as well as the physical environment. Lastly, the multiple, and often unanticipated, effects of targeted gene disruption on aggressive behavior are considered.