内侧前额叶皮质DNA甲基化调控大鼠酒精相关行为

酒精滥用不仅导致组织器官损伤,还易诱发神经精神疾病。研究表明,DNA甲基化在酒精诱导基因表达和行为改变中发挥重要作用,但具体的神经生物学机制尚未被阐明。为了探索DNA甲基化在酒精滥用中的作用机制,本研究选取健康成年雄性SD大鼠(Rattus norvegicus)32只,随机分为饮水对照组(n=16)和慢性酒精暴露组(n=16),运用双瓶选择实验(two bottle choice test,TBCT)评估大鼠酒精偏爱率(alcohol preference),通过旷场行为(open field test,OFT)评估活动状态并检测血酒精浓度。分离两组大鼠内侧前额叶皮质(medial prefrontal cortex,mPFC),提取总DNA,利用简化代表性重亚硫酸盐测序技术(reduced representation bisulfite sequencing,RRBS)构建mPFC甲基化谱,对差异基因进行功能富集和通路分析,筛选与酒精滥用密切相关的甲基化差异基因,运用qRT-PCR技术检测差异基因的表达,验证DNA甲基化对基因的表达调控;利用qRT-PCR和Western blot检测甲基转移酶(DNA methyltransferases,DNMTs)和甲基化CpG位点结合蛋白2(methyl CpG binding protein 2,MeCP2)的表达;同时,还检测了短期酒精暴露(7 d)对大鼠mPFC内DNMTs和MeCP2的影响(n=8/组)。结果表明,慢性酒精暴露大鼠mPFC内基因启动子区甲基化水平显著升高。与酒精滥用密切相关的差异基因中,慢性酒精暴露组Ntf3和Ppm1G启动子区甲基化水平升高,mRNA表达降低;Hap1和DUSP1启动子区甲基化水平降低,mRNA表达升高。慢性酒精暴露使DNMT3B和MeCP2 mRNA和蛋白表达升高,而短期内酒精暴露不影响它们的表达。本研究初步证实DNA甲基化与酒精滥用的发展相关,可能受DNMT3B和MeCP2分子的调控,并发现了与酒精滥用相关的靶基因Ntf3、Ppm1G、Hap1和DUSP1,为研究酒精滥用的神经生物学机制提供了新见解,同时为酒精滥用治疗提供了可能的药理学靶点。     

Phenylephrine enhances glutamate release in the medial prefrontal cortex through interaction with N‐type Ca2+ channels and release machinery

α₁‐adrenoceptors (α₁‐ARs) stimulation has been found to enhance excitatory processes in many brain regions. A recent study in our laboratory showed that α₁‐ARs stimulation enhances glutamatergic transmission via both pre‐ and post‐synaptic mechanisms in layer V/VI pyramidal cells of the rat medial prefrontal cortex (mPFC). However, a number of pre‐synaptic mechanisms may contribute to α₁‐ARs‐induced enhancement of glutamate release. In this study, we blocked the possible post‐synaptic action mediated by α₁‐ARs to investigate how α₁‐ARs activation regulates pre‐synaptic glutamate release in layer V/VI pyramidal neurons of mPFC. We found that the α₁‐ARs agonist phenylephrine (Phe) induced a significant enhancement of glutamatergic transmission. The Phe‐induced potentiation was mediated by enhancing pre‐synaptic glutamate release probability and increasing the number of release vesicles via a protein kinase C‐dependent pathway. The mechanisms of Phe‐induced potentiation included interaction with both glutamate release machinery and N‐type Ca²⁺ channels, probably via a pre‐synaptic G_q/phospholipase C/protein kinase C pathway. Our results may provide a cellular and molecular mechanism that helps explain α₁‐ARs‐mediated influence on PFC cognitive functions.

     

电刺激大鼠内侧额叶前皮质对听皮层神经元频率感受野可塑性的调制

本文应用常规电生理学技术,研究电刺激大鼠内侧额叶前皮质（medial prefrontal cortex,mPFC）对初级听皮层神经元频率感受野（receptive field,RF）可塑性的调制。电刺激mPFC,137个听皮层神经元（72．8％）RF可塑性受到影响,其中抑制性调制71个神经元（37．7％）,易化性调制66个神经元（35．1％）,其余51个神经元（27．2％）不受影响。mPFC的抑制性调制效应表现为,RF的偏移时间延长,恢复时间缩短。相反,mPFC的易化性调制效应表现为,RF的偏移时间缩短,恢复时间延长。电刺激mPFC对RF可塑性的调制与声、电刺激之间的时间间隔有关,最佳时间间隔介于5-30ms之间。结果提示,大鼠mPFC可以调制听皮层神经元的功能活动,可能参与听觉学习记忆过程。     

Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex

Rats raised in an enriched environmental condition (EC) exhibit a decreased (35%) maximal velocity (V(max)) of [3H]dopamine (DA) uptake in medial prefrontal cortex (mPFC) compared with rats raised in an impoverished condition (IC); however, no differences between EC and IC groups in V(max) for [3H]DA uptake were found in nucleus accumbens and striatum. Using biotinylation and immunoblotting techniques, the present study examined whether the brain region-specific decrease in DA transporter (DAT) function is the result of a reduction in DAT cell surface expression. In mPFC, nucleus accumbens and striatum, total DAT immunoreactivity was not different between EC and IC groups. Whereas no differences in cell surface expression of DAT were found in nucleus accumbens and striatum, DAT immunoreactivity in the biotinylated cell surface fraction of mPFC was decreased (39%) in EC compared with IC rats, consistent with the magnitude of the previously observed decrease in V(max) for [3H]DA uptake in mPFC in EC rats. These results suggest that the decrease in DAT cell surface expression in the mPFC may be responsible for decreased DAT function in the mPFC of EC compared with IC rats, and that there is plasticity in the regulatory mechanisms mediating DAT trafficking and function.     

Oxytocin suppresses basal glutamatergic transmission but facilitates activity-dependent synaptic potentiation in the medial prefrontal cortex

Both oxytocin and oxytocin receptors are implicated in neuropsychiatric disorders, particularly autism which involves a severe deficit in social cognition. Consistently, oxytocin enhances social cognition in humans and animals. The infralimbic medial prefrontal cortex (IL-mPFC) is believed to play an important role in the regulation of social cognition which might involve top-down control of subcortical structures including the amygdala. However, little is known about whether and how oxytocin modulates synaptic function in the IL-mPFC. The effect of oxytocin on excitatory neurotransmission in the IL-mPFC was studied by examining both the evoked and spontaneous excitatory neurotransmission in the IL-mPFC layer V pyramidal neurons before and after perfusion with oxytocin. To investigate the effect of oxytocin on synaptic plasticity, low-frequency stimulation-induced long-lasting depression was studied in oxytocin-treated brain slices. Oxytocin produced a significant suppression of glutamatergic neurotransmission in the IL-mPFC layer V pyramidal neurons which was mediated by a reduction in glutamate release. Activation of the cannabinoid CB1 receptors was involved in this pre-synaptic effect. Treatment of brain slices with oxytocin for 1?h converted long-lasting depression into long-lasting potentiation of glutamatergic neurotransmission. This oxytocin-mediated plasticity was NMDA receptor-dependent and was mediated by the synaptic insertion of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors. The aforementioned suppression of basal glutamatergic neurotransmission and facilitation of activity-dependent synaptic plasticity in the IL-mPFC might be critical for the effect of oxytocin on social cognition.     

Nicotine increases dopamine clearance in medial prefrontal cortex in rats raised in an enriched environment

Environmental enrichment results in differential behavioral and neurochemical responsiveness to nicotine. The present study investigates dopamine clearance (CL_DA) in striatum and medial prefrontal cortex (mPFC) using in vivo voltammetry in rats raised in enriched (EC) or impoverished conditions (IC) and administered nicotine (0.4 mg/kg) or saline. Baseline CL_DA in striatum or mPFC was not different between EC and IC. Across repeated DA application, striatal CL_DA increased in saline-control EC and IC. CL_DA increased in mPFC in saline-control IC; CL_DA did not change in saline-control EC. Thus, enrichment differentially alters dynamic responses of the dopamine transporter (DAT) to repeated DA application in mPFC, but not in striatum. In EC, nicotine increased mPFC CL_DA compared to saline-control, but had no effect on CL_DA in IC; nicotine had no effect in striatum in EC or IC. Compared to respective saline-controls, nicotine increased dihydroxyphenylacetic acid content in striatum and mPFC in EC, but not in IC. Nicotine also had no effect on DA content in striatum or mPFC in EC or IC. Results indicate that enrichment eliminated the dynamic response of mPFC DAT to repeated DA application in saline-control and augmented the nicotine-induced increase in DAT function in mPFC, but not in striatum.     

Role of medial prefrontal cortex dopamine in age differences in response to amphetamine in rats: Locomotor activity after intra-mPFC injections of dopaminergic ligands

Changes in medial prefrontal cortex (mPFC) dopamine receptor expression and in mPFC projections to the nucleus accumbens in adolescence suggest that there may be age differences in the regulation of drug‐related behavior by the mPFC. The age‐specific role of prelimbic D1 dopamine receptors on amphetamine‐induced locomotor activity was investigated. In experiment 1, rats aged postnatal day 30 (P30), P45, and P75, corresponding to early and late adolescence and adulthood, were given an injection of D1 and D2 antagonists into the prelimbic mPFC before a systemic injection of 1.5 mg/kg of amphetamine and locomotor activity was recorded. In experiment 2, effects of intra‐prelimbic injections of a D1 agonist and antagonist on locomotor activity produced by a lower dose (0.5 mg/kg) of amphetamine were investigated. D2 receptor antagonist did not alter amphetamine‐induced activity, whereas the D1 receptor antagonist reduced activity produced by 1.5 mg/kg of amphetamine more in P30 than in P45 and P75 rats. In addition, D1 agonist enhanced the locomotor activating effects of 0.5 mg/kg of amphetamine in adolescent rats and decreased activity in adult rats. These results suggest that insufficient activation of mPFC D1 receptors may underlie the reduced activity at the low dose of amphetamine in early adolescent compared to adult rats. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

创伤后应激障碍大鼠前额内侧皮质MR表达的变化

目的观察创伤后应激障碍(PTSD)样大鼠前额内侧皮质(medial prefrontal cortex,mPFC)神经元核受体-盐皮质激素受体(Mineralocorticoid receptors,MR)表达的变化。方法采用国际认定的单一连续应激(single prolonged stress,SPS)方法建立PTSD大鼠模型,取成年健康雄性Wistar大鼠90只,随机分为PTSD模型1d、7d、14d、28d和正常对照组。采用免疫组化、免疫印迹和RT-PCR方法分别进行各组mPFC神经元MR表达变化的观察及检测,进行图像分析和统计学处理。结果 PTSD大鼠mPFC神经元MR的表达在SPS-1d时高于对照组,随后下降,SPS-14d最低,SPS-28d恢复性上调,但仍然低于对照组(P<0.05)。结论 PTSD模型大鼠经SPS处理后,mPFC中出现MR表达的变化,该变化可能参与PTSD的下丘脑-垂体-肾上腺(hypothalamic pituitary adren axis,HPA)轴的变化机制。     

Mineralocorticoid receptors in the medial prefrontal cortex and hippocampus mediate rats' unconditioned fear behaviour

Corticosterone is released from the adrenal cortex in response to stress, and binds to glucocorticosteroid receptors (GRs) and mineralocorticosteroid receptors (MRs) in the brain. Areas such as the dorsal hippocampus (DH), ventral hippocampus (VH) and medial prefrontal cortex (mPFC) all contain MRs and have been previously implicated in fear and/or memory.The purpose of the following experiments was to examine the role of these distinct populations of MRs in rats’ unconditioned fear and fear memory.The MR antagonist (RU28318) was microinfused into the DH, VH, or mPFC of rats. Ten minutes later, their unconditioned fear was tested in the elevated plus-maze and the shock-probe tests, two behavioral models of rat “anxiety.” Twenty-four hours later, conditioned fear of a non-electrified probe was assessed in rats re-exposed the shock-probe apparatus.Microinfusions of RU28318 into each of the three brain areas reduced unconditioned fear in the shock-probe burying test, but only microinfusions into the VH reduced unconditioned fear in the plus-maze test. RU28318 did not affect conditioned fear of the shock-probe 24 hr later.MRs in all three areas of the brain mediated unconditioned fear to a punctate, painful stimulus (probe shock). However, only MRs in the ventral hippocampus seemed to mediate unconditioned fear of the more diffuse threat of open spaces (open arms of the plus maze). In spite of the known roles of the hippocampus in spatial memory and conditioned fear memory, MRs within these sites did not appear to mediate memory of the shock-probe.     

Age‐dependent expression of Nav1.9 channels in medial prefrontal cortex pyramidal neurons in rats

Developmental changes that occur in the prefrontal cortex during adolescence alter behavior. These behavioral alterations likely stem from changes in prefrontal cortex neuronal activity, which may depend on the properties and expression of ion channels. Nav1.9 sodium channels conduct a Na⁺ current that is TTX resistant with a low threshold and noninactivating over time. The purpose of this study was to assess the presence of Nav1.9 channels in medial prefrontal cortex (mPFC) layer II and V pyramidal neurons in young (20‐day old), late adolescent (60‐day old), and adult (6‐ to 7‐month old) rats. First, we demonstrated that layer II and V mPFC pyramidal neurons in slices obtained from young rats exhibited a TTX‐resistant, low‐threshold, noninactivating, and voltage‐dependent Na⁺ current. The mRNA expression of the SCN11a gene (which encodes the Nav1.9 channel) in mPFC tissue was significantly higher in young rats than in late adolescent and adult rats. Nav1.9 protein was immunofluorescently labeled in mPFC cells in slices and analyzed via confocal microscopy. Nav1.9 immunolabeling was present in layer II and V mPFC pyramidal neurons and was more prominent in the neurons of young rats than in the neurons of late adolescent and adult rats. We conclude that Nav1.9 channels are expressed in layer II and V mPFC pyramidal neurons and that Nav1.9 protein expression in the mPFC pyramidal neurons of late adolescent and adult rats is lower than that in the neurons of young rats. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1371–1384, 2017     

In vivo evidence that constitutive activity of serotonin2C receptors in the medial prefrontal cortex participates in the control of dopamine release in the rat nucleus accumbens: differential effects of inverse agonist versus antagonist

Control of the mesoaccumbens dopamine (DA) pathway by central serotonin_2C receptors (5-HT_2CRs) involves different 5-HT_2CR populations located within multiple brain areas. Here, using in vivo microdialysis in halothane-anesthetized rats, we assessed the role of medial prefrontal cortex (mPFC) 5-HT_2CRs in the control of basal and activated accumbal DA outflow, to identify the modalities of their recruitment and the role of 5-HT_2CR constitutive activity. Intra-mPFC injection of the 5-HT_2CR inverse agonist SB 206553 (0.5 μg/0.2 μL), without effect by itself, decreased accumbal DA outflow induced by morphine (2.5–10 mg/kg, s.c.), haloperidol (0.01 mg/kg, s.c.) or GBR 12909 (2.5 mg/kg, i.p.). Conversely, intra-mPFC injection of the 5-HT_2CR antagonist SB 242084 (0.5 μg/0.2 μL), without effect by itself, decreased the effect of 10 mg/kg morphine, the only drug enhancing basal 5-HT outflow in the mPFC. The inhibitory effect of SB 206553 on 2.5 mg/kg morphine-stimulated DA outflow was suppressed by the concomitant intra-mPFC injection of SB 242084. Finally, changes of basal DA outflow induced by the 5-HT_2CR agonist Ro 60-0175 (3 mg/kg, i.p.) or SB 206553 (5 mg/kg, i.p.) were unaffected by intra-mPFC injection of SB 242084. These results, showing that 5-HT_2CR antagonist and inverse agonist behave differently in vivo, demonstrate that mPFC 5-HT_2CRs facilitate activated accumbal DA outflow and that 5-HT_2CR constitutive activity participates in this interaction.     

Antipsychotic drugs reverse the AMPA receptor-stimulated release of 5-HT in the medial prefrontal cortex

The prefrontal cortex (PFC) is involved in the pathophysiology of schizophrenia. PFC neuronal activity is modulated by monoaminergic receptors for which antipsychotic drugs display moderate-high affinity, such as 5-HT(2A) and alpha(1)-adrenoceptors. Conversely, PFC pyramidal neurons project to and modulate the activity of raphe serotonergic neurons and serotonin (5-HT) release. Under the working hypothesis that atypical antipsychotic drugs may partly exert their action in PFC, we assessed their action on the in vivo 5-HT release evoked by increasing glutamatergic transmission in rat medial PFC (mPFC). This was achieved by applying S-AMPA in mPFC (reverse dialysis) or by disinhibiting thalamic excitatory afferents to mPFC with bicuculline. The application of haloperidol, chlorpromazine, clozapine and olanzapine in mPFC by reverse dialysis (but not reboxetine or diazepam) reversed the S-AMPA-evoked local 5-HT release. Likewise, the local (in mPFC) or systemic administration of these antipsychotic drugs reversed the increased prefrontal 5-HT release produced by thalamic disinhibition. These effects were shared by the 5-HT(2A) receptor antagonist M100907 and the alpha(1)-adrenoceptor antagonist prazosin. However, raclopride (DA D2 antagonist) had very modest effects. These results suggest that, besides their action in limbic striatum, antipsychotic drugs may attenuate glutamatergic transmission in PFC, possibly by interacting with 5-HT(2A) and/or alpha(1)-adrenoceptors.     

Bidirectional variation in glutamate efflux in the medial prefrontal cortex induced by selective positive and negative allosteric mGluR5 modulators

Dysregulation of prefrontal cortical glutamatergic signalling via NMDA receptor hypofunction has been implicated in cognitive dysfunction and impaired inhibitory control in such neuropsychiatric disorders as schizophrenia, attention‐deficit hyperactivity disorder and drug addiction. Although NMDA receptors functionally interact with metabotropic glutamate receptor 5 (mGluR5), the consequence of this interaction for glutamate release in the prefrontal cortex (PFC) remains unknown. We therefore investigated the effects of positive and negative allosteric mGluR5 modulation on changes in extracellular glutamate efflux in the medial PFC (mPFC) induced by systemic administration of the non‐competitive NMDA receptor antagonist dizocilpine (or MK801) in rats. Extracellular glutamate efflux was measured following systemic administration of the positive allosteric mGluR5 modulator [S‐(4‐Fluoro‐phenyl)‐{3‐[3‐(4‐fluoro‐phenyl)‐[1,2,4]‐oxadiazol‐5‐yl]‐piperidin‐1‐yl}‐methanone] (ADX47273; 100 mg/kg, p.o.) and negative allosteric mGluR5 modulator [2‐chloro‐4‐{[1‐(4‐fluorophenyl)‐2,5‐dimethyl‐1H‐imidazol‐4‐yl]ethynyl}pyridine] (RO4917523; 0.3 mg/kg, p.o.), using a wireless glutamate biosensor in awake, freely moving rats. The effect of MK801 (0.03–0.06 mg/kg, s.c.) on mPFC glutamate efflux was also investigated in addition to the effects of MK801 (0.03 mg/kg, s.c.) following ADX47273 (100 mg/kg, p.o.) pre‐treatment. ADX47273 produced a sustained increase in glutamate efflux and increased the effect of NMDA receptor antagonism on glutamate efflux in the mPFC. In contrast, negative allosteric mGluR5 modulation with RO4917523 decreased glutamate efflux in the mPFC. These findings indicate that positive and negative allosteric mGluR5 modulators produce long lasting and opposing actions on extracellular glutamate efflux in the mPFC. Positive and negative allosteric modulators of mGluR5 may therefore be viable therapeutic agents to correct abnormalities in glutamatergic signalling present in a range of neuropsychiatric disorders.

     

Mechanical stimulation evokes rapid increases in extracellular adenosine concentration in the prefrontal cortex

Mechanical perturbations can release ATP, which is broken down to adenosine. In this work, we used carbon‐fiber microelectrodes and fast‐scan cyclic voltammetry to measure mechanically stimulated adenosine in the brain by lowering the electrode 50 μm. Mechanical stimulation evoked adenosine in vivo (average: 3.3 ± 0.6 μM) and in brain slices (average: 0.8 ± 0.1 μM) in the prefrontal cortex. The release was transient, lasting 18 ± 2 s. Lowering a 15‐μm‐diameter glass pipette near the carbon‐fiber microelectrode produced similar results as lowering the actual microelectrode. However, applying a small puff of artificial cerebral spinal fluid was not sufficient to evoke adenosine. Multiple stimulations within a 50‐μm region of a slice did not significantly change over time or damage cells. Chelating calcium with EDTA or blocking sodium channels with tetrodotoxin significantly decreased mechanically evoked adenosine, signifying that the release is activity dependent. An alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate receptor antagonist, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, did not affect mechanically stimulated adenosine; however, the nucleoside triphosphate diphosphohydrolase 1,2 and 3 (NTDPase) inhibitor POM‐1 significantly reduced adenosine so a portion of adenosine is dependent on extracellular ATP metabolism. Thus, mechanical perturbations from inserting a probe in the brain cause rapid, transient adenosine signaling which might be neuroprotective.

     

Abnormal medial prefrontal cortex connectivity and defective fear extinction in the presymptomatic G93A SOD1 mouse model of ALS

Amyotrophic lateral sclerosis (ALS) is a fatal progressive neuropathy associated with the degeneration of spinal and brainstem motor neurons. Although ALS is essentially considered as a lower motor neuron disease, prefrontal cortex atrophy underlying executive function deficits have been extensively reported in ALS patients. Here, we examine whether prefrontal cortex neuronal abnormalities and related cognitive impairments are present in presymptomatic G93A Cu/Zn superoxide dismutase mice, a mouse model for familial ALS. Structural characteristics of prelimbic/infralimbic (PL/IL) medial prefrontal cortex (mPFC) neurons were studied in 3-month-old G93A and wild-type mice with the Golgi–Cox method, while mPFC-related cognitive operations were assessed using the conditioned fear extinction paradigm. Sholl analysis performed on the dendritic material showed a reduction in dendrite length and branch nodes on basal dendrites of PL/IL neurons in G93A mice. Spine density was also decreased on basal dendrite segments of branch order five. Consistent with the altered morphology of PL/IL cortical regions, G93A mice showed impaired extinction of conditioned fear. Our findings indicate that abnormal prefrontal cortex connectivity and function are appreciable before the onset of motor disturbances in this model.     

Stimulation of group I mGlu receptors in the ventrotegmental area enhances extracellular dopamine in the rat medial prefrontal cortex

Group I mGlu receptors have been implicated in the control of brain dopamine release. However, the receptor subtype involved and the precise site of action have not been determined. In this study we show that (R,S)3,5-dihydroxyphenylglycine (DHPG; 6 and 60 nmol ICV), a selective group I mGlu receptor agonist, raised extracellular dopamine respectively by 176% and 243% of basal values in the medial prefrontal cortex as assessed by in vivo microdialysis in conscious rats. (R,S)2-chloro-5-hydroxyphenylglycine (60 nmol ICV), a selective mGlu5 receptor agonist, raised extracellular dopamine by 396% of basal values. Intra-VTA DHPG (0.6–6 nmol) mimicked ICV injection whereas intracortical infusion (1–1000 µmol/L) had no effect. DHPG-induced rise of extracellular dopamine was reversed by tetrodotoxin and by the selective mGlu1 and mGlu5 receptor antagonists 7(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate (CPCCOEt) and 2-methyl-6-(phenylethynyl)pyridine (MPEP) either ICV or into the ventrotegmental area (VTA), suggesting that neuronal release and both mGlu1 and mGlu5 receptors were involved. These results support the existence of functional mGlu1 and mGlu5 receptors in the VTA regulating the release of dopamine in the medial prefrontal cortex.     

The (in)complete organelle genome: exploring the use and nonuse of available technologies for characterizing mitochondrial and plastid chromosomes

Not long ago, scientists paid dearly in time, money and skill for every nucleotide that they sequenced. Today, DNA sequencing technologies epitomize the slogan ‘faster, easier, cheaper and more’, and in many ways, sequencing an entire genome has become routine, even for the smallest laboratory groups. This is especially true for mitochondrial and plastid genomes. Given their relatively small sizes and high copy numbers per cell, organelle DNAs are currently among the most highly sequenced kind of chromosome. But accurately characterizing an organelle genome and the information it encodes can require much more than DNA sequencing and bioinformatics analyses. Organelle genomes can be surprisingly complex and can exhibit convoluted and unconventional modes of gene expression. Unravelling this complexity can demand a wide assortment of experiments, from pulsed‐field gel electrophoresis to Southern and Northern blots to RNA analyses. Here, we show that it is exactly these types of ‘complementary’ analyses that are often lacking from contemporary organelle genome papers, particularly short ‘genome announcement’ articles. Consequently, crucial and interesting features of organelle chromosomes are going undescribed, which could ultimately lead to a poor understanding and even a misrepresentation of these genomes and the genes they express. High‐throughput sequencing and bioinformatics have made it easy to sequence and assemble entire chromosomes, but they should not be used as a substitute for or at the expense of other types of genomic characterization methods.