DNA-barcoding is a rapidly developing method for efficiently identifying samples to species level by means of short standard DNA sequences. However, reliable species assignment requires the availability of a comprehensive DNA barcode reference library, and hence numerous initiatives aim at generating such barcode databases for particular taxa or geographic regions. Historical museum collections represent a potentially invaluable source for the DNA-barcoding of many taxa. This is particularly true for birds and mammals, for which collecting fresh (voucher) material is often very difficult to (nearly) impossible due to the special animal welfare and conservation regulations that apply to vertebrates in general, and birds and mammals in particular. Moreover, even great efforts might not guarantee sufficiently complete sampling of fresh material in a short period of time. DNA extracted from historical samples is usually degraded, such that only short fragments can be amplified, rendering the recovery of the barcoding region as a single fragment impossible. Here, we present a new set of primers that allows the efficient amplification and sequencing of the entire barcoding region in most higher taxa of Central European birds and mammals in six overlapping fragments, thus greatly increasing the value of historical museum collections for generating DNA barcode reference libraries. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens. DNA-barcoding, the sequencing of standardized, species-specific parts of the genome , facilitHence, historical museum collections, accumulated over decades to centuries and expertly identified and curated, represent an extremely valuable source of tissue for molecular studies in general  and DNA-Despite the widely acknowledged value of historical museum collections for DNA-barcoding projects, most DNA-barcoding studies published to date have mainly relied on fresh material obtained via extensive fieldwork, because DNA in museum specimens tends to degrade within a few years, resulting in often limited DNA quantity and quality. Both natural postmortem processes and customary preservation methods cause degradation of DNA –18, suchA ~650 bp region at the 5’ end of the mitochondrial cytochrome c oxidase subunit 1 gene (COI) is commonly used as the DNA-barcoding region for most animal taxa, including vertebrates [Apart from dead animals collected under a permit issued by the provincial government of Styria, only catalogued museum samples or pieces of meat  bought at a supermarket  were used in this study. Therefore, no further permits were needed.www.boldystems.org)  barcoding region in Central European birds and mammals in six overlapping fragments were designed based on alignments of full DNA barcodes of representatives of all bird and mammal orders occurring in Central Europe, downloaded from BOLD (ems.org) . To get ems.org)  based onTGAC-3’; ) to allowww.abol.ac.at; [A total of 69 tissue samples from frozen or ethanol-preserved material  was obtained from dead specimens collected in the framework of the Austrian Barcode of Life (ABOL) project , from mTotal genomic DNA of frozen or ethanol-preserved material was extracted by means of a rapid Chelex protocol . DNA ext® Taq DNA Polymerase (Invitrogen) and 1–2 μl or 5 μl of DNA extract of fresh (Chelex extraction) or museum (Kit extraction) samples, respectively. PCR conditions followed the manufacturer’s protocol for the polymerase used. We employed a two-step PCR protocol with an initial denaturation step at 94°C for 2 minutes, followed by 8 cycles at 94°C for 30 seconds, 46 or 48°C for 30 seconds and 1 minute at 72°C, plus another 27 cycles at 94°C for 30 seconds, 48 or 50°C for 30 seconds and 1 minute at 72°C  following the manufacturer's instructions. Purified PCR products were sequenced bidirectionally in 10 μl sequencing reactions using the BigDye® Sequence Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), applying the protocol described in [Amplification via polymerase chain reaction was conducted in 25 μl PCR reactions containing 1X PCR Buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM primer, 2 U Platinumribed in . Sequencwww.boldsystems.org) and are publicly available in the project titled ‘ABOL—museum primers , MPBM’.DNA sequences were aligned in MEGA 6.06 . AmbiguoPipistrellus nathusii). Primer pair A_int2 amplified the majority of fresh bird samples as well as all fresh mammal samples, except for cattle (Bos taurus), black rat (Rattus rattus) and the majority of bats. Samples that did not amplify with this primer pair worked well with either primer pair A_int2_2 or M_int2 (Myotis nattereri) those few samples that did not work with this primer pair could be amplified with A_int4, which worked well in all birds. In birds, the last fragment was amplified either with primer pair end_A1 or end_A2, or in the case of the great crested grebe (Podiceps cristatus) with end_M3. In mammals, this region proved to be more difficult to amplify, and various primer pairs were employed to amplify it (again with the exception of some bat species).We designed and tested sets of 5’-M13-tagged PCR primers that reliably amplify the DNA-barcoding region in short (108–183 bp) overlapping DNA fragments in Central European bird and mammals orders , Table 2Bombycilla garrulus), for which no fragments could be amplified, probably due to insufficient DNA amount or quality, amplification and sequencing success was high in the stuffed hide samples, with only single fragments lacking in European green woodpecker (Picus viridis) and European mole . Two regions failed to amplify in the barn owl . Contrary to the high amplification and sequencing success of stuffed hide samples, none of the tanned hide samples worked, with the exception of the raccoon (Procyon lotor), for which all except the fourth fragment were successfully amplified and sequenced. As all fragments were amplified in fresh/ethanol preserved European mole and raccoon samples and representatives of the Piciformes and Strigiformes  and 14 tanned hide  samples. The age of these specimens ranged from 2 to 61 years , whereas zero or one substitution was observed in most other species. For the muskrat (Ondatra zibethicus) specimen NMW 68327, we obtained barcodes from both ethanol preserved tissue and the stuffed hide, and both sequences were identical.For some species, two or three samples were barcoded. The maximum number of substitutions observed within a species was three in the roe deer (Martes foina), amplified with primer pair M_int4, no internal stop codons were observed, and BLAST searches in GenBank resulted in clear matches with published COI sequences, confirming that we have not sequenced mitochondrial pseudogene copies integrated into the nuclear genome which appear to be quite common in mammals and birds [With the exception of the fourth internal region in stone martens  approaches for barcoding both fresh and historical material, significantly reducing time and costs for conducting DNA-barcoding studies –43. ThusBarcoding historical bird and mammal specimens has long been a tedious and time-consuming task, prohibiting their use in large-scale barcoding projects. This study established a new primer set for amplifying the DNA-barcoding region in Central European birds and mammals in six short overlapping fragments, which allows for the efficient barcoding of the many treasures present in museum collections, such that barcoding activities need not rely solely on fresh, frozen or ethanol-preserved material. Applying our new primer set in recently established NGS protocols promises to further increase the efficiency of barcoding old bird and mammal specimens. Nonetheless, unless a project requires the inclusion of type material—which would be beneficial for barcoding projects in any case–or taxa for which no fresh material is available, we advocate the use of fresh material whenever possible.S1 Data(FAS)Click here for additional data file.S1 FigThe only sample that worked was the positive control, an ethanol-preserved European hare sample (NMW 69075). For more information on samples  see (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Ih current amplitudes are reduced in layer (L) 5 pyramidal neurons of rats with freeze lesion induced malformations. These changes were associated with an increased EPSP temporal summation. Here, we examine the effects of HCN channel inhibition on synaptic responses in fast spiking, presumptive basket cells and accommodating, presumptive Martinotti, GABAergic interneurons in slices from freeze lesioned animals. In control animals, fast spiking cells showed small sag responses which were reduced by the HCN channel antagonist ZD7288. Fast spiking cells in lesioned animals showed absent or reduced sag responses. The amplitude of single evoked EPSPs in fast spiking cells in the control group was not affected by HCN channel inhibition with ZD7288. EPSP ratios during short stimulus trains at 25 Hz were not significantly different between control and lesion groups. ZD7288 produced an increase in EPSP ratios in the control but not lesion groups. Under voltage clamp conditions, ZD7288 did not affect EPSC ratios. In the control group, accommodating interneurons showed robust sag responses which were significantly reduced by ZD7288. HCN channel inhibition increased EPSP ratios and area in controls but not the lesioned group. The results indicate that HCN channels differentially modulate EPSPs in different classes of GABAergic interneurons and that this control is reduced in malformed rat neocortex.Cortical malformations are often associated with pharmaco-resistant epilepsy. Alterations in hyperpolarization-activated, cyclic nucleotide-gated, non-specific cation (HCN) channels have been shown to contribute to malformation associated hyperexcitability. We have recently demonstrated that expression of HCN channels and  Ih, a non-inactivating, cationic current activated by membrane hyperpolarization  channels  cutting solution consisting of (in mM): 135 N-Methyl-D-glucamine, 1.5 KCl, 1.5 KH2PO4, 23 choline HCO3, 0.4 ascorbic acid, 0.5 CaCl2, 3.5 MgCl2 and 25 D-glucose  with oxygenated recording solution kept at 32 ± 1°C.Rats were anesthetized with isoflurane and decapitated. The brain was quickly removed and placed in ice-cold oxygenated  microscope equipped with Nomarski optics or a Zeiss Axio Examiner D1  microscope equipped with Dodt contrast optics, each equipped with a 40×-water immersion lens and infrared illumination. L5 interneurons were identified by their somatic shape, size, absence of a prominent apical dendrite, distance from the pial surface, intrinsic properties and spiking properties.A receptors were inhibited with bicuculline methiodide  in all experiments. Synaptic responses were evoked with a concentric bipolar stimulating electrode  or twisted pair nichrome bipolar electrode positioned within several hundred micrometers of the recorded interneuron. Single EPSPs were evoked at a rate of 0.1 Hz with 100 μs pulses 20–200 μA in amplitude. Trains of five EPSPs at 25 Hz were used to examine synaptic integration. Traces shown are the average of 5–10 consecutive responses.Whole cell recordings were obtained from visually and physiologically identified cells in L5 of the neocortex. Signals were acquired with either an Axopatch 200B amplifier  or a ELC-03XS npi bridge balance amplifier  controlled by Clampex software via a Digidata 1322A or 1550A interface (Molecular Devices). Responses were digitized at 5 kHz, and analyzed offline with Clampfit software. Patch electrodes with an open tip resistance of 2–5 MΩ were pulled from borosilicate glass tubes. Tight seals (>1 GΩ) were obtained between the patch electrodes and the neurons before using suction to break into whole cell mode. Only recordings with a series resistance <25 MΩ were used for the study and recordings in which a >20% increase in series resistance was observed were excluded. Patch electrodes were filled with an internal solution containing (in MM): 125 K-gluconate, 10 KCl, 10 HEPES, 2 Mg-ATP, 0.2 Na-GTP, 0.5 EGTA, and had an adjusted pH and osmolarity of 7.3 and 290, respectively. GABAt-test, using paired t-tests for analysis of data acquired before and after drug application, or with a two-way ANOVA. Student’s t-tests were one-tailed unless otherwise noted. P < 0.05 was considered significant.Data is either shown as dots representing individual data points or as means ± the standard error. Statistical analysis was carried out with either a Students Sag responses were evaluated using hyperpolarizing current injection in 50 pA intervals. Sag amplitude was calculated as the maximum membrane hyperpolarization following current onset minus the steady state potential prior to offset of the hyperpolarizing current injection. Synaptic integration was evaluated using EPSP ratios calculated as the percent change in the amplitude of the fifth evoked event relative to the amplitude of the first event. The total area under the curve of evoked trains was normalized to the amplitude of the first EPSP to account for potential changes in input as stimulus intensity was kept constant for pre- and post-drug trials.Drugs were stored in frozen stock solution and dissolved in the recording solution prior to each experiment. BIC was present at all times in all conditions. After recording control responses, ZD7288  was washed in for 10 min to block HCN channels.GABAergic interneurons serve to regulate network activity . The responses to hyperpolarizing current injection before and after bath application of ZD7288 for a L5 basket cell in the hyperexcitable zone of a lesioned animal are shown in Figure n = 16, 2-way ANOVA, p < 0.05). Overall, the sag responses to a 250 pA negative current step were significantly reduced in interneurons from freeze lesioned rats compared to controls . Rebound responses were virtually absent in both groups . These results are consistent with decreased HCN channel functioning in L5 fast spiking cells in lesioned animals.We obtained somatic, whole-cell patch clamp recordings from L5 interneurons in neocortical slices from control and lesioned animals. Recordings in lesioned animals were obtained in the hyperexcitable zone 1–2 mm lateral to the lesion  and input resistance  of a population of interneurons from control and lesioned animals were not significantly different  or lesioned group  and lesioned groups . Similar responses from a fast spiking neuron in the lesion group are shown in Figure n = 13; ZD7288: 2.4 ± 0.5 mV, n = 13; paired t-test, p > 0.05).HCN channel inhibition has been reported to increase EPSP amplitude and temporal summation in L5 pyramidal neurons . This is consistent with our observation of minimal sag responses in controls and virtual absence of such responses in the lesion group. It has been reported that HCN channel inhibition also results in an increase in EPSP area  or lesion group . Under voltage-clamp conditions, evoked EPSCs did not exhibit significant summation in cells from control or lesioned animals  or lesioned groups . No difference was seen in baseline EPSC ratios between interneurons from control and lesion animals . ZD7288 had no significant effect on EPSC area in either the control  or lesion group . These results suggest that the observed effects of HCN channel inhibition on EPSP area in fast spiking interneurons are postsynaptically mediated.EPSCs were also examined in interneurons voltage clamped at −60 mV to minimize  Figures . HCN chan = 10, 2-way ANOVA, p < 0.001; Lesion: n = 11, 2-way ANOVA, p < 0.0001). Rebound responses were also observed in these cells at the offset of hyperpolarizing current steps. Application of ZD7288 caused a significant reduction of the rebound amplitude in control and lesion groups . As shown in Figure n = 11, Lesion: n = 11; 2-way ANOVA, p = 0.24). In control animals, significantly larger sag  and rebound responses  were observed in accommodating cells compared to fast-spiking cells (data not shown). The resting membrane potentials of accommodating interneurons in the control and lesion groups were not significantly different  and lesion groups  or lesion animals . Analysis of EPSP areas showed that ZD7288 increased the area significantly in the control group .The above results indicate that L5 accommodating interneurons have HCN channel mediated sag responses and changes in RMP and input resistance consistent with the presence of an Ih activation  or lesion group . Under voltage-clamp conditions, evoked EPSC ratios were variable in interneurons from both control and lesion animals  or lesion groups  or lesion group  cells and accommodating (presumed SOM positive) neurons and that properties of these GABAergic interneurons are altered in malformed cortex.Immunocytochemical studies have documented HCN channel expression in the dendrites of neocortical and hippocampal pyramidal cells with a pronounced increase in density as a function of distance from the soma  cells and the fast-spiking PV-expressing basket cells. SOM-positive cells display ascending axon collaterals which reach L1 and ramify extensively (Kawaguchi and Kubota, The RMP and input resistance of fast-spiking interneurons in control and lesioned groups were not significantly different. HCN channel inhibition did not significantly affect the RMP in either group whereas input resistance was increased in both. HCN channel mediated sag responses elicited by hyperpolarizing current pulses were small and had an atypical time course in the control group. In the lesion group, sag responses were absent or significantly reduced. Responses in both control and lesion groups were significantly reduced by ZD7288. Overall, this complex pattern of changes is consistent with the presence of HCN channels capable of influencing input resistance but not RMP. The location of these channels is unclear as is the factors responsible for the unusual time course of sag responses.Ih currents. We have previously shown that HCN mediated responses are reduced in L5 pyramidal neurons in freeze lesioned rats (Albertson et al., In the fast spiking cells, inhibition of HCN channels with ZD7288 did not significantly affect singly-evoked EPSPs in interneurons from control or lesioned groups. EPSP area during short trains of stimulation was increased in the control group after pharmacological inhibition of HCN channels whereas increased area was not observed in the lesion group. This is consistent with a loss of HCN modulation of EPSPs in the lesion group. When neurons were voltage clamped at −60 mV, no effect of ZD7288 on EPSC area was observed in either the control or lesion group, suggesting that HCN channels modulate excitatory inputs onto L5 basket cells via a postsynaptic mechanism. In the present study, we demonstrated that L5 fast-spiking cells had small atypical responses to hyperpolarizing current pulses, implying the existence of small, possible remote HCN-mediated Interneurons with accommodating firing patterns are presumptive SOM-positive cells. The RMP of accommodating interneurons in control and lesioned groups were not significantly different whereas input resistance was significantly higher in the lesion group. RMP was hyperpolarized in the presence of ZD7288 by a small but significant amount in the control but not the lesion group. Input resistance was significantly increased in by groups by HCN channel inhibition. In keeping with previous studies (Ma et al., HCN channel inhibition increased EPSP ratios and area in controls but not the lesioned group. This suggests that HCN channels play a significant role in controlling synaptic excitability of accommodating interneurons, a role that is disrupted in malformed cortex. The functional significance of this for seizures needs to be established. The differential effects on EPSP ratios and sag responses in the lesion group could potentially be due to the observed increases in input resistance or differential effects on somatic and dendritic HCN channels.Ih in individual circuit elements including interneurons is essential for understanding how the loss of HCN channels alters network excitability. Imbalance of excitation and inhibition contributes to aberrant network activity (Yizhar et al., Defining the role of One alternative mechanism is increased presynaptic release of glutamate following HCN channel inhibition. HCN channels have previously been implicated in the control of GABA release. In the cerebellum, basket cell terminals show immunogold labeling for HCN channels (Luján et al., In summary, HCN channel inhibition alters intrinsic and synaptic properties of GABAergic interneurons. We observed an increase in EPSP ratios in accommodating interneurons following HCN channel inhibition suggesting that GABA release could be increased. However, the increase was not as robust as that observed previously in pyramidal cells. The functional consequences of HCN loss may differ between excitatory and inhibitory cell types. These data suggest that while HCN channels may serve a similar dendritic filtering role in some interneurons, HCN channel influence in pyramidal neurons is greater. We also did not observe any evidence of presynaptic HCN channels at excitatory synapses onto fast spiking L5 interneurons.AJA, ASB and JJH designed the research and wrote the manuscript. AJA and ASB conducted experiments and analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Arabidopsis thaliana communities. Plant-derived signals released by the wounded plant resulted in more elaborate root development in the neighboring, unwounded plants. Such plant-derived signals also upregulated the Aluminum-activated malate transporter (ALMT1) responsible for the secretion of malic acid (MA) and the DR5 promoter, an auxin responsive promoter concentrated in root apex of the neighboring plants. We speculate that plant-derived signal-induced upregulation of root-specific ALMT1 in the undamaged neighboring plants sharing the environment with stressed plants may associate more with the benign microbes belowground. We also observed increased association of beneficial bacterium Bacillus subtilis UD1022 on roots of the neighboring plants sharing environment with the damaged plants. Wounding-induced plant-derived signals therefore induce defense mechanisms in the undamaged, local plants, eliciting a two-pronged preemptive response of more rapid root growth and up-regulation of ALMT1, resulting in increased association with beneficial microbiome.When disrupted by stimuli such as herbivory, pathogenic infection, or mechanical wounding, plants secrete signals such as root exudates and volatile organic compounds (VOCs). The emission of VOCs induces a response in the neighboring plant communities and can improve plant fitness by alerting nearby plants of an impending threat and prompting them to alter their physiology for defensive purposes. In this study, we investigated the role of plant-derived signals, released as a result of mechanical wounding, that may play a role in intraspecific communication between  ALMT1), which has been shown to also be activated in response to other abiotic responses  from Arabidopsis thaliana roots, which augments recruitment of the beneficial rhizobacterium Bacillus subtilis UD1022-a plant–microbial interaction that decreases susceptibility to many foliar pathogens  and a benign (Sinorhizobium meliloti) microbe trigger secretions of different proteins, indicating plants use different chemicals to signal different neighbors (Arabidopsis plant grown in larger monocultures produce more defense metabolites (glucosinolates) compared to smaller monocultures . These transgenic reporter lines offer insight on two belowground, induced-defense mechanisms observed in unwounded plants exposed in close proximity to injured neighbors. The triggered defense responses include the upregulation of the ALMT1 gene and an auxin-responsive DR5 gene, and accelerated lateral and primary root growth. We report an unusual shoot-to-root interplant communication leading to altered belowground root responses and benign biotic associations.In the present study, we speculated that plants sharing the space with a mechanically injured neighbor may show differences in root plasticity. We also questioned how the recipient community perceives damaged-derived chemical signals and evaluated its impact on root growth and root–microbe interactions. The current study relies on measurements of root growth rate and fluorescence assays using the β-glucuronidase (GUS) reporter gene in two transgenic reporter lines of Arabidopsis thaliana ecotype Columbia (Col-0) were obtained from the Arabidopsis Biological Resource Center (ABRC) and surface sterilized using 50% sodium hypochlorite for 1 min and then thrice washed with sterile water. ALMT1::GUS and DR5::GUS transgenic lines were obtained from Hiroyuki Koyama  and Wendy Peer (University of Maryland). The seeds were cultured on Murashige and Skoog (MS)  or partition petri plates (with one seedling on each side of the partition).Seeds of wild-type oog (MS)  solid agALMT1::GUS, and DR5::GUS assays, 12-days-old seedlings were transferred from solid MS media to 6-well culture plates (Fisher Scientific) containing 2.5 mL of 0.5x MS liquid medium with 0.05 mM MES and 3% sucrose. Each 6-well plate contained two seedlings, placed in corner wells opposite and diagonal from one another to maximize distance apart. Plants were grown for 12 days with constant shaking at 90 rpm.For root colonization, Arabidopsis plant. In both the Col-0, and ALMT1::GUS assay experiments, one seedling in each petri plate was designated the “Donor” community and was mechanically wounded, while its adjacent seedling was left untouched. Mechanical wounding occurred on the same day as seedling transfer. Non-invasive (non-puncturing) contact of the forceps on the seedlings established control trials in which neither seedling was wounded. Primary root growth rate was measured and calculated as μm h-1 over 8 days post-wounding.Sterilized, room-temperature forceps created 4 distinct punctures to the lamina of 2 of the first true leaves on each “donor” ® film (Bemis) divide agar but allow for shared airspace. The plates used for the ALMT1::GUS assay were half-filled with a solid MS agar with 3% sucrose, while the other halves of the plates were filled with MS agar with 10 μM AlCl3 (Sigma-Aldrich). Two seedlings, 8-days post-germination, were transferred to the plates and allowed to grow per the earlier growth conditions. Mechanical wounding of the randomly selected “donor” seedling (that which “donates” VOCs) occurred on the same day as transfer. Eight days after transfer, the unwounded seedling in each plate was processed per the published description of the β-Glucuronidase Reporter Gene Staining assay (Sigma-Aldrich) and stored at 4°C in a 4% formaldehyde solution until microscopy on an AxioCam color dissecting microscope.Partition plates sealed with Parafilm MBacillus subtilis UD1022 was streaked from a -80°C glycerol stock onto a plate of low-salt Luria Bertani (LB) medium  and grown for 24 h at 28°C with shaking at 180 RPM. A subculture was started in 200 mL of LB liquid culture from the previous streak. After shaking for 24 h at 28°C, the subculture was diluted 1:1000 and incubated further at 28°C. When the subculture OD600 reached 0.6–1.0, 10 μL of inoculum (OD600 = 0.007 of UD1022) were added to the existing 0.5X MS liquid medium in the wells of the “recipient” A. thaliana plants.®13  and 1:500 concentration Calcofluor (Sigma-Aldrich), which were left in contact with the roots for 20 ± 3 min and then rinsed once with sterile water. Images were captured with a 25X C-Apochromat objective on a Zeiss LSM 710. Spectral data was collected on the 710 spectral detector. Collected spectral data was used in online fingerprinting and images were post-processed channel unmixed resulting in blue , green (SYTO®13 in UD1022 biofilm) and red (auto-fluorescence) layers. Limited amounts of SYTO®13 are suspected to have penetrated root vascular tissue and cause increased green fluorescence outside of the UD1022 biofilm.Adherent UD1022 cells and biofilm on root surface were imaged using laser scanning confocal microscopy. After 24 h shaking at 90 rpm and 6 h stationary at 21 ± 2°C under the photoperiod described for growth conditions, UD1022-inoculated plants were removed from media and roots were sliced from aboveground plant body. Root samples were then placed in sterile 1-mL tubes (Eppendorf), rinsed once with Phosphate Buffer (2.5 mM), and then suspended in 1-mL of buffer. Histological staining relied on 1:1000 concentration STYOTwo seedlings, 15-days post-germination, were transferred to partition plates with a solid MS agar with 3% sucrose (Sigma-Aldrich) and allowed to grow per the earlier growth conditions. Mechanical wounding of the randomly selected “donor” seedling occurred on the same day as transfer. Five days after transfer, the unwounded seedling in each plate was processed per the published description of the β-Glucuronidase Reporter Gene Staining assay and stored at 4°C in a 4% formaldehyde solution until microscopy on an AxioCam color dissecting microscope.® Pro, Version 11 . When necessary to compare two means, Student’s two-tailed t-test were also generated using JMP® Pro, Version 11.The data were analyzed by a one-way analysis of variance (ANOVA) using JMPA. thaliana plant facilitated the release of airborne VOCs that induced an elaborate series of defense-mechanisms in the neighboring seedlings. The VOCs upregulated the root-specific malate transporter (ALMT1) gene, increasing recruitment of a beneficial bacterium, and the DR5 auxin promoter, which accelerated the root growth.Mechanical wounding of Figures 1A,C). The Recipient (the unwounded seedling) and Donor (the seedling that is wounded and releases VOCs) seedlings were positioned either at 4 or 2 cm apart from each other, in an effort to identify any changes in response that may be due to weakened potency of the VOC signal over greater distance between plants. We used partition- and no-partition- prototypes to check if the Donor community releases both VOCs and root exudates to trigger change in phenotype in the Recipient communities . We speculated that the no other signals besides VOCs could be exchanged in the partition plates, hence VOCs may play a critical role in signaling between injured donors and recipient communities. Mechanical wounding of the Donor seedling was followed by the observation that, at 4 cm apart, the primary lateral roots of Recipients next to wounded Donors grew significantly faster at 224 ± 63 μm h-1 than the 137 ± 29 μm h-1 of the seedlings next to unwounded Donors. There was, however, no significant difference in the growth rate of primary lateral root (“PR”) between the 2 and 4 cm spaced trials . This result necessitates further investigation into the potency of the VOC signal over larger distance. The number of lateral root extensions (“LR”) on the Recipient seedlings was also counted, with Recipient communities next to wounded Donors showing an average of 4.25 more lateral roots than Recipients with unwounded Donors .We designed a prototype to test if wounding neighboring plants changes belowground phenotype in the neighboring communities . Recipient plants next to wounded Donors exhibited accelerated root growth consistent with that observed in the undivided petri plates: the mean primary root growth rate was 220 ± 9 μm h-1 compared to the control growth rate of 164 ± 11 μm h-1 . A strong causational pattern is established here between exposure to the VOCs elicited by mechanical wounding and acceleration of growth in PR and LR, suggesting a commensalistic relationship in which wounded plants signal potentially vulnerable neighboring plants of the same species to mitigate damage by increasing biomass.Partition plates were used to assess whether the noted VOC signal functioned as a diffusion signal and would still affect the Recipient community without sharing the same medium . Blue staining was concentrated in gradients toward root apex of the primary and lateral root extensions. The significant difference in the magnitude of gradient in the GUS staining suggests that a signaling cascade for the DR5 auxin reporter is triggered by the VOCs documented in this study; the DR5 upregulation is consistent with the earlier observation that primary root and lateral root extensions were longer and more numerous in our earlier experiments.Both Donor and Recipient groups in the wounded treatment exhibited longer PR and a greater number of LR than the control Donor and Recipient groups, suggesting that the VOCs released by mechanical wounding may upregulate the auxin response that results in increased accumulation of auxin in the apical meristem of primary roots in both the Recipient and Donor plants. To confirm the involvement of auxin interplay in the VOC triggered Recipient communities, we used an auxin reporter ALMT1) play a vital role in shaping root microbiome in plants infected with an aboveground pathogen . GUS expression was concentrated in lateral root extensions and the apical meristem of the primary root, suggesting activation of malate transporter by wounded-induced VOCs.Previously, we have shown that root specific malate transporters . Post 24 h of exposure to the wounded/unwounded plants’ VOCs, Recipient communities were checked for B. subtilis colonization and biofilm formation using confocal microscopy. Confocal imaging of Recipient plant roots exposed to the wounded Donors revealed significantly more UD1022 biofilm development than existent on control roots (exposed to the unwounded Donors) inoculated with identical levels of UD1022 . This result suggests that malate upregulation induced by wounded Donors may also associate more with the beneficial bacterium UD1022.Having shown that wound-induced VOCs trigger malate transporter expression in the Recipient communities, we tested whether the increased malate transporter expression triggered more root colonization by benign bacterium The impact of aboveground to belowground signaling in intra-plant communications is a field that has recently gained a lot of attention. Both biotic and abiotic stress trigger mobile signals between the aerial and root parts of a plant  Various DR5 gene marked with the GUS reporter gene in this study is a synthetic auxin-responsive promoter that indicates high auxin accumulation  seen in the Recipients in proximity to wounded Donors. The increase in the auxin-responsive promoter DR5 in roots of neighboring plants exposed to the damaged neighbors suggests that auxin activity operates differently under interplant wound signaling compared to intraplant wound signaling.Wounding response at the intraplant level is very well-characterized. It has been shown that wounded plants trigger both local and systemic responses at the intra-plant level . It has mulation . In our ALMT1 expression followed by increased colonization by UD1022. The matrix of cells  surrounding only the Recipient root next to a wounded neighbor illustrates the increased recruitment of B. subtilis associated with the upregulation of ALMT1. Previous research has shown that this strain is an effective plant growth-promoting rhizobacteria and reduces foliar entry of deleterious pathogens (Our work adds a layer of complexity to the previously documented above- and belowground interactions involving VOCs and microbiome interactions. Recipient plants exposed to damaged neighbors showed increased athogens . It is aathogens . The litathogens . Conversathogens . We haveathogens , but we athogens . It woulA. thaliana evolved to anticipate abiotic and biotic stress and survived most when root systems matured at a faster rate, allowing for adequate nutrient and moisture uptake even in potentially contaminated soil.In this study, we report that aboveground mechanical wounding elicits substantial belowground changes in plant phenotypic and genotypic characteristics. Previous literature has shown the defense-catalyzing capabilities of VOCs on intraplant and interspecific systems . LikewisALMT1 malate transporter in increasing biofilm development and the DR5 auxin reporter in accelerating root growth. These findings contribute to a growing body of research on root–microbe interactions and expand the agricultural applications of VOCs as factors for pathogen protection and plant growth promotion. Our study demonstrates that the volatiles released by damaged plants elicit belowground changes related to root plasticity and root–microbe interactions in neighboring plants under controlled conditions. Many questions still remain regarding the capabilities of this specific aboveground-belowground VOC relationship: how concentrated does the VOC signal have to be to effectively upregulate auxin-responsive and malate transporter genes under natural conditions? What is the specific chemical composition of the VOC signal at play in this interplant interaction? What other genotypic transductions do these VOCs cause, aside from the two genes of interest reported in the present study? Do VOCs derived from wounded plants play a role in belowground interspecies signaling? The answers to these inquiries ultimately march closer to a new breed of organic crop primers that secure more robust, disease-free crop yields without relying on unsustainable industrial fertilizers.Our hypothesis that the same defense response elicited by VOCs in intraplant and interspecific systems would be induced between neighboring, but anatomically separate plants was correct. We identified the benefits of the CS conducted all the experiments described in the manuscript. VL and CS analyzed the data. CS performed the statistics on the root growth analysis. CS and HB drafted the manuscript. HB conceived the study and CS participated in its design and coordination. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In vivo imaging studies indicate an increased presynaptic dopamine synthesis capacity in striatal terminals and cell bodies in the midbrain in schizophrenia; however, measures of the dopamine-synthesising enzyme, tyrosine hydroxylase (TH), have not identified consistent changes. We hypothesise that dopamine dysregulation in schizophrenia could result from changes in expression of dopamine synthesis enzymes, receptors, transporters or catabolic enzymes. Gene expression of 12 dopamine-related molecules was examined in post-mortem midbrain (28 antipsychotic-treated schizophrenia cases/29 controls) using quantitative PCR. TH and the synaptic dopamine transporter (DAT) proteins were examined in post-mortem midbrain (26 antipsychotic-treated schizophrenia cases per 27 controls) using immunoblotting. TH and aromatic acid decarboxylase (AADC) mRNA and TH protein were unchanged in the midbrain in schizophrenia compared with controls. Dopamine receptor D2 short, vesicular monoamine transporter (VMAT2) and DAT mRNAs were significantly decreased in schizophrenia, with no change in DRD3 mRNA, DRD3nf mRNA and DAT protein between diagnostic groups. However, DAT protein was significantly increased in putatively treatment-resistant cases of schizophrenia compared to putatively treatment-responsive cases. Midbrain monoamine oxidase A (MAOA) mRNA was increased, whereas MAOB and catechol-O-methyl transferase mRNAs were unchanged in schizophrenia. We conclude that, whereas some mRNA changes are consistent with increased dopamine action (decreased DAT mRNA), others suggest reduced dopamine action (increased MAOA mRNA) in the midbrain in schizophrenia. Here, we identify a molecular signature of dopamine dysregulation in the midbrain in schizophrenia that mainly includes gene expression changes of molecules involved in dopamine synthesis and in regulating the time course of dopamine action.The dopamine hypothesis of schizophrenia posits that increased subcortical dopamine underpins psychosis.  Two meta-analyses of 11 and 15 imaging studies, respectively, found significant elevations in dopamine synthesis capacity in the striatum in schizophrenia,10 and a further imaging study identified increased dopamine synthesis capacity in the midbrain.11 Here, we ask whether molecular evidence of presynaptic dopamine changes can be found in the substantia nigra in people with schizophrenia.Subcortical dopamine dysregulation is considered the final common pathway in the pathophysiology of schizophrenia.L-tyrosine to L-DOPA, which is then converted by aromatic acid decarboxylase (AADC) to dopamine.7 TH is regulated at transcriptional, translational and post-translational levels12 and, although in rodents electroconvulsive shock treatment does not change midbrain TH mRNA levels,13 regulation of midbrain TH mRNA does occur in response to pharmacological agents.15 Studies measuring TH in the midbrain of people with schizophrenia do not concur, with no change,17 increases18 and region-specific decreases19 reported in people with schizophrenia compared with controls. Although previously no change was found in AADC mRNA in the midbrain of schizophrenia patients,16 positron-emission tomography (PET) imaging studies have suggested increases in AADC activity in schizophrenia patients.11 Thus, further studies of TH and AADC within the basal ganglia are needed to determine whether molecular changes consistent with increased dopamine biosynthesis occur in schizophrenia.Dopamine neurotransmission has multiple steps of possible regulation. Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, converts 20 DRD2 and DRD3 isoforms are expressed in the dopamine neuron somatodendritic field in the substantia nigra and VTA and at presynaptic terminals.21 Presynaptic DRD2 and DRD3 full-length (referred to as DRD3) are autoreceptors, inhibiting dopamine neuron firing, dopamine release and terminating dopamine synthesis.26 Alternatively, spliced DRD3 variants that produce truncated proteins that do not bind dopamine have been identified, the most abundant being DRD3 non-functional (DRD3nf).28 DRD3nf modulates dopamine reception by forming heteromers with DRD2 and DRD3, redirecting dopamine-binding receptors into an intracellular compartment and thus reducing DRD3 and DRD2 cell surface action.29 DRD3 mRNA is decreased, whereas DRD3nf mRNA is increased in the cortex  of schizophrenia patients30- setting a precedent for testing whether altered DRD3 expression occurs in the midbrain in schizophrenia.Dopamine interacts with five G-protein-coupled receptors, dopamine receptor (DR) D2, DRD3 and DRD4 (inhibitory D2-like family) as well as DRD1 and DRD5 (excitatory D1-like family).31 However, gene expression of DRD2 isoforms, DRD3 or DRD3nf within the midbrain in schizophrenia, has not yet been examined.We hypothesised that changes in mRNA encoding presynaptic dopamine receptors may contribute to changes in dopamine regulation in the midbrain in schizophrenia. A study of post-mortem midbrain showed increased DRD2 receptor binding in substantia nigra from schizophrenia cases.32 and these molecules also represent a possible site of dysregulation in schizophrenia. The ability of psychoactive drugs to bring about psychotic-like states by binding to DAT or VMAT2  imply that alterations in dopamine transport can contribute to psychosis. Dopamine is catabolised in dopamine neurons and glial cells by monoamine oxidases (MAOs) and catechol-O-methyl transferase (COMT).36 Midbrain monoamine oxidase A (MAOA) and MAOB are expressed in human substantia nigra,37 and inhibition of MAOs contributes to amphetamine-induced psychosis.33 Thus, decreased synthesis of dopamine breakdown or reduced dopamine transport proteins in schizophrenia may contribute to hyperdopaminergia or to dopamine dysregulation.Dopamine action is terminated by reuptake from the synaptic cleft via the dopamine transporter (DAT) and dopamine is packaged into vesicles for release by vesicular monoamine transporter 2 (VMAT2),We ask whether gene expression of 12 dopamine-related molecules and/or protein expression of TH and DAT are changed in the substantia nigra from individuals who suffered from chronic schizophrenia compared with healthy individuals. We hypothesised that TH and AADC mRNA would be increased in the midbrain in schizophrenia with a corresponding increase in TH protein in the midbrain. Further, we hypothesised that DAT mRNA and protein would be decreased in the midbrain of people with schizophrenia compared with controls. We hypothesised that gene expression of other molecules involved in dopamine reception, transport or catabolism may contribute to dysregulation of dopamine neurotransmission, and thus may be changed in the midbrain in schizophrenia compared with controls.α=0.05)38 and tissue availability. Substantia nigra was excised from midbrain cryostat-generated 60 μm slices based on TH immunolabelling of adjacent 14 μm slide-mounted sections . Hemisected fresh frozen midbrain tissue neuroanatomically matched at the level of the oculomotor nerve from 30 schizophrenia cases and 30 control individuals (14 μm sections mounted on gelatin-coated glass slides and adjacent 60 μm sections collected between wax paper) were provided by the New South Wales Brain Tissue Resource Centre. Sample size was selected based on previous post-mortem studies  was used as an indicator of possible treatment resistance versus all other antipsychotics at time of death (n=21–19). Antipsychotic medication was converted to chlorpromazine (CPZ) equivalents 42 (n=22) or more prevalent negative symptoms (n=7) with one unknown. Toxicology screening of control cases at time of post-mortem revealed nothing (n=11), diazepam (n=1), cannabis (n=1), paracetamol (n=7), codeine (n=3) and blood pressure medication (n=2), and some (n=5) had no toxicology screen with cardiac failure recorded as cause of death. Toxicology screening of schizophrenia cases revealed no comorbid substance use (n=13), methadone and diazepam (n=1), diazepam and insulin (n=1), diazepam, pethidine, codeine and paracetamol (n=1), codeine, diazepam and morphine (n=1), paracetamol only (n=4), a non-steroidal anti-inflammatory (n=1), morphine (n=1) codeine (n=1) and ibuprofen (n=1) and screening not available (n=5). Alcohol consumption at time of death in controls and schizophrenia cases was as follows: nil (n=1–0), <20 g per day (n=17–22), 20–50 g per day (n=6–3), 50–80 g per day (n=1–0), >80 g per day (1–3) and unknown (n=4–2). A history of depression symptoms during lifetime was identified in eight schizophrenia cases (six were treated with serotonin-selective reuptake inhibitors (SSRIs) and two with tricyclic antidepressants) and in one control  and the final midbrain protein cohort comprised 26 schizophrenia cases and 27 controls . In both cohorts, diagnostic groups were matched for age, gender and post-mortem interval (PMI), and in the mRNA cohort diagnostic groups were matched for RNA integrity number (RIN)39 . All sch dose)42 . The sch43 Frozen slides (14 μm) were thawed at room temperature and fixed in 4% paraformaldehyde in phosphate-buffered saline . After rinsing, endogenous peroxidase activity was blocked using 30% MeOH and 3% H2O2 (20 min), and slides were rinsed again and blocked with 10% normal horse serum  for 1 h. Anti-TH mouse primary antibody was applied overnight at 4 °C at a 1:1000 dilution . After rinsing, the slides were incubated with a horse anti-mouse IgG-biotinylated secondary antibody  at a 1:500 dilution  followed by rinsing and incubation with the avidin–biotin–peroxidase complex  and then application of 3,3′-diaminobenzidine solution  to visualize TH immunoreactivity (2 min). The slides were rinsed, dehydrated, stained with Nissl (5 min exposure to 0.02% thionin) and cover-slipped. Slides were visualised on a Nikon Eclipse 80i light microscope. TH immunohistochemistry was scored  based on overall TH staining intensity (including cell bodies and fibres) for the area of the midbrain equivalent to the area excised for homogenisation  and TaqMan gene expression assays . The following inventoried Taqman assays were used to measure the gene expression of three housekeeper genes . The following inventoried TaqMan assays were used to measure gene expression of dopamine-related mRNAs: TH, Hs00165941; AADC, Hs01105048; MAOA, Hs00165140; MAOB, Hs00168533; COMT, Hs00241349; DAT, Hs00997364; VMAT2, Hs00996835; DRD2S, Hs01014210, DRD3, Hs00949496 and DRD3nf, Hs00945868. Custom probes were designed for DRD2L (AIHSPG8) and DRD2longer (AII1NNG) based on published sequences.45Total RNA from substantia nigra samples was extracted with Trizol . RNA quality was determined using the Agilent Bioanalyzer 2100  and samples with low RIN were removed from gene expression analysis (one control and two schizophrenia cases). Complementary DNA was synthesised using the Superscript III First Strand Synthesis Kit (Life Technologies). Quantitative real-time PCR (qPCR) analysis was conducted as reported previously46Serial dilutions of pooled complementary DNA from all samples were included on every qPCR plate for quantitation of sample expression by the relative standard curve method. All qPCR reactions were performed in triplicate. The SDS 2.4 software  was employed to analyse the qPCR data. Expression levels of the triplicate means of sample expression were normalised to the geomean of the housekeepers.47 Midbrain samples were homogenised (0.1 M Tris (pH 7.5), 50% glycerol, proteinase inhibitor cocktail and aprotinin (0.015 mM), all Sigma-Aldrich) using a handheld electric homogeniser . Proteins were quantified using the Bradford protein assay (Sigma-Aldrich). An aliquot of each sample was pooled and run in duplicate on all gels for standardisation between immunoblots . Standard curves (0.5–15 μg protein) were run to determine the linear range of expression. Protein concentrations loaded for TH and DAT detection for two western runs/protein were 3 and 10 μg per sample for TH and DAT, respectively. Proteins were separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose membranes . Membranes were blocked in 5% non-fat milk  and incubated in primary antibody overnight at 4 °C. Primary antibodies were mouse anti-TH  and rabbit anti-DAT . Secondary antibodies were horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit . Bands were visualised using chemiluminescence  and captured on a Chemidoc XRS system (Bio-Rad). Band densities were determined using Quantity One Software  and expressed numerically as quantity values (mm × intensity). All membranes were re-probed with mouse anti-β-actin  as a loading control.Western blotting was performed as previously described.χ2-test was used to determine the relationship between diagnosis and TH intensity scores determined from immunohistochemistry. The Grubb’s test  was used to exclude group outliers  in genes or proteins of interest after normalising to housekeepers. Independent-sample two-tailed t-tests were used to test for differences in mRNA and protein cohort demographics . A  and RIN ) and indThe relative intensity of protein bands  was divided by the internal control for standardisation between blots. The relative intensity of TH and DAT bands was then normalised to the corresponding β-actin band relative intensity and tested for normality. To achieve a normal distribution, TH and DAT protein data underwent log10 transformations. The levels of the mRNAs of interest were normalised to the geometric mean (referred to as geomean3) of the housekeeper mRNAs  and the relative gene expression of each gene was tested for normality (Shapiro–Wilk test). Data that were not normally distributed were transformed. Monoamine oxidase and DRD3nf mRNA underwent log10 transformation and TH mRNA underwent a square root transformation.If normality was not achieved after transformation, as in the case of COMT mRNA, the non-normally distributed mRNA data were analysed using the Mann–Whitney test. Spearman’s correlations for COMT mRNA were performed with continuous demographic variables .t-test was used to detect diagnostic differences.For all normally distributed mRNA and protein measures, Pearson’s correlations were performed with continuous demographic variables ). For all mRNA and protein measures, Spearman’s correlations were conducted with antipsychotic drug measurements and duration of illness in the schizophrenia group. If a correlation was detected, analysis of covariance was used to test for diagnostic differences between controls and schizophrenia cases. Factorial analysis of variance was used to determine the effect of smoking status at time of death and agonal state on gene and protein expression in the diagnostic groups. If an effect was detected, this was included as a fixed factor in the analysis of covariance. If no correlations with demographic variables or effect of smoking or agonal state were detected, an independent sample two-tailed P⩽0.05.Relative gene expression and relative protein expression data were converted to a percentage of the control group mean  and graphed as mean±s.e.m. Statistical significance was set at t-tests were used to explore differences in gene or protein expression in the schizophrenia group based on antipsychotic treatment, symptoms and depression, and statistical significance was set at P⩽0.05. We highlight that this analysis is exploratory as the group sizes are modest and no corrections are made for multiple analyses.The effect of antipsychotics on gene and protein expression was explored in three ways. First, correlation analyses of mRNAs and proteins with CPZ equivalents (described above) were performed. Second, comparisons between schizophrenia cases divided into those treated with mostly first-generation antipsychotics and those treated with mostly second-generation antipsychotics. Third, comparisons were made between cases using clozapine at time of death (possible treatment resistance) versus those on antipsychotics other than clozapine at time of death, although we acknowledge that not taking clozapine does not rule out treatment resistance. In addition, the effect of mostly positive or mostly negative symptoms and presence of depression symptoms during lifetime or no depression symptoms were explored in the schizophrenia cases. Student’s two-tailed The relationships between demographic variables were assessed, and detailed statistical data are shown in r=0.458, P<0.0001, n=57) and gene expression of all three housekeepers individually. None of the housekeeper genes or the geomean3 correlated with PMI. Brain pH and PMI were not correlated with relative intensity of β-actin protein bands. All housekeeper genes and geomean3  showed strong positive correlations with RNA quality as determined by RIN, in the full cohort and by diagnostic group.The relationship between demographic variables and housekeeper mRNAs and β-actin protein were assessed. Detailed statistical analysis is reported in Most genes of interest in the mRNA expression cohort positively correlated with both RIN and pH, except for DRD2longer, which only correlated with RIN and AADC, DRD3nf and DRD3 mRNAs, which only correlated with pH . Only THP=0.395) levels in the substantia nigra between control and schizophrenia brains were identified by qPCR  intensity of TH immunolabeling in the substantia nigra and diagnosis . A TH and β-actin band were detected at the expected molecular weights, ~59 and ~42 kDa, respectively, in all samples (P=0.584) was detected by western blotting .No significant difference in TH mRNA  in substantia nigra from schizophrenia relative to controls; however, this difference did not reach statistical significance , DRD2L mRNA by 29%  and DRD2Longer mRNA by 22% .Although DRD3 was decreased by 26.76% and DRD3nf was decreased by 23.20% in substantia nigra of people with schizophrenia compared with controls, neither decrease reached statistical significance , but no diagnostic changes were found in the levels of MAOB mRNA  or COMT mRNA  and also a significant 37% decrease in the levels of VMAT2 mRNA  in the substantia nigra of schizophrenia cases when compared with controls . No significant difference in DAT protein expression was detected between control and schizophrenia . In contrast, we detected a 72.5% increase in DAT protein expression in cases on clozapine compared with cases treated with other antipsychotics . There were no changes in gene or protein expression when schizophrenia cases that received mostly first-generation antipsychotics were compared with schizophrenia cases that received mostly second-generation antipsychotics .None of the dopamine-related genes or proteins of interest measured in the substantia nigra from schizophrenia cases correlated significantly with measures of antipsychotic use or duration of illness from either the mRNA or protein expression cohorts . In addit=−2.188, df=20, P=0.041). There were no changes in any other gene or protein expression based on symptoms. No gene or protein of interest varied significantly in the schizophrenia group based on the presence of depression symptoms within their lifetime (data not shown).When schizophrenia cases displaying mostly positive or negative symptoms were compared, there was a significant 56.5% increase in DRD3 mRNA in the primarily positive symptoms group compared with those people with schizophrenia displaying mostly negative symptoms 50 are important for TH level and activity. Midbrain TH mRNA and protein levels were highly correlated, thus suggesting that our measurements of human TH are accurate and valid. However, we measured only one TH mRNA transcript (pan) and four TH mRNA transcripts have been identified in human brain.52 Thus, changes in individual TH transcripts and their corresponding protein isoforms may have been missed in our study. Additionally, our study did not compare subregions (VTA versus substantia nigra) or dorsal or ventral distribution within these regions and a recent study identified reductions in TH protein in subregions of the midbrain in schizophrenia.19Whereas midbrain TH mRNA and protein levels were unchanged between schizophrenia and controls, we observed a greater range of values in schizophrenia compared with controls in both mRNA and protein, perhaps reflecting the contradictory evidence for changes (both increases and decreases) found in previous studies.53 Thus, increased dopamine synthesis capacity via AADC appears to be changed as a consequence of schizophrenia and may not be only secondary to antipsychotic treatment. In contrast, PET imaging studies in schizophrenia patients, who were antipsychotic-naive or had not received treatment in the previous 6 months and subsequently treated subchronically (20–45 days) with haloperidol (a first-generation antipsychotic with high affinity for DRD2), showed a downregulation of this AADC-dependent step.54 Contrary to our hypothesis, but in line with the study by Grunder et al.,54 we found a trend for a decrease (22.9%) in AADC mRNA levels in the midbrain of patients with schizophrenia that suggests that there could be less synthesis of AADC in chronic, medicated patients. In rodent whole-brain homogenates, haloperidol and loxapine treatments increased AADC mRNA, whereas the second-generation antipsychotic sulpiride (a DRD2 and DRD3 antagonist) did not.55 The differential effects of distinct antipsychotics on AADC gene expression may reduce the ability to detect a consistent change in AADC in post-mortem tissue and exposure to antipsychotics may mask potential changes in AADC in schizophrenia. It is proposed that some schizophrenia patients do not respond to antipsychotic treatment as they do not exhibit the elevation in dopamine synthesis capacity that is typically associated with the disorder.56 However, using clozapine at the time of death as a proxy for possible treatment resistance40 did not reveal a difference in AADC gene expression. Interestingly, there was an indication of a negative correlation with the last CPZ equivalent dose and AADC mRNA, suggesting that in our study, the AADC mRNA reductions may be due to antipsychotic treatment.Increased activity of the second step in the dopamine synthesis pathway has been identified in schizophrenia. PET studies measuring 18F-DOPA uptake and conversion to 18F-dopamine show that the dopamine biosynthetic step that is AADC-dependent is elevated in people at ultrahigh risk for psychosis.58 however, imaging studies indicated no change in striatal DAT binding in schizophrenia patients.60 Similar binding studies have not been carried out in the midbrain of schizophrenia patients in whom dopamine can be released in the somatodendritic field to regulate dopamine neuron activity via feedback inhibition. Further work including study of DAT protein, binding and activity, both in post-mortem and PET imaging studies in the brains of people with schizophrenia  compared with controls, are needed to more fully characterize the anatomical sites of DAT abnormalities, especially in the midbrain, and to better understand the implications of our current findings. This is, however, to our knowledge, the first study to implicate reduced midbrain DAT gene expression in the pathophysiology of schizophrenia, highlighting a putative major dysregulation of DAT.As a major finding of this study, we found a 45% decrease in DAT mRNA in schizophrenia. This may seem at odds with our lack of ability to detect a change in DAT protein levels in the substantia nigra in schizophrenia. The lack of diagnostic change in DAT protein could suggest that DAT mRNA changes may not have an impact on protein levels in midbrain or that the protein function/stability may be altered causing feedback changes on DAT transcription. Alternatively, there may be increased translation of DAT protein or decreased DAT utilisation and breakdown, either of which could result in no change in steady-state DAT protein levels with a decrease in DAT mRNA. However, we report a decrease in DAT protein in cases treated with antipsychotics other than clozapine  relative to those treated with clozapine —such differences may contribute to the difficulty in detecting a diagnostic change in DAT protein expression overall and suggest that posttranscriptional regulation of DAT may vary with clinical state. Alternatively, it is possible that the decrease in DAT mRNA may be reflected in DAT protein changes in either the dorsal or ventral striatum or in cortical sites, which were not examined in this study. As dopamine action in the striatum is primarily terminated by dopamine reuptake from the synaptic cleft by DAT, less DAT levels or DAT action leading to slower termination of dopamine neurotransmission could contribute to hyperdopaminergia in schizophrenia. In the striatum, post-mortem studies report reduced levels of DAT binding in schizophrenia;61 A decrease in VMAT2 gene expression in the substantia nigra may contribute to reduced VMAT2 protein and suggests less efficient vesicular packaging of dopamine; however, less VMAT2 action can contribute to hyperdopaminergia in certain contexts as inhibition of VMAT2 contributes to amphetamine-induced psychosis.34 In contrast to our study, VMAT2 binding was found to be increased in the ventral brainstem in a PET imaging study in schizophrenia;61 however, this apparent difference may be due to regional differences in the expression of dopamine molecules in the brainstem. These observations set the stage for designing experimental systems that would mimic the putative state of transporter abnormalities found in schizophrenia and could serve as a platform for testing the impact of novel therapies.In addition to a reduction in the mRNA encoding the main dopamine transporter localized to the outer cell membrane, we also find reduced mRNA encoding dopamine transporter localized to the vesicles  in schizophrenia, VMAT2 mRNA does not appear to be regulated by antipsychotics in our study or in others.62 Serotonin released in the substantia nigra by dorsal raphe serotonergic fibres results in activation of 5-HT2 receptors and subsequent inhibition of dopamine neuron firing.63 Therefore, more MAOA synthesis/activity in the midbrain in schizophrenia could lead to increased serotonin breakdown and reduced inhibitory modulation of dopamine neurons by serotonin, thus contributing to the dopamine dysregulation. Increased cortical COMT activity, inferred from genetic changes, was linked to increased midbrain TH mRNA in normal subjects;64 however, we find no change in midbrain COMT mRNA levels in people with schizophrenia compared with controls, supporting previous studies in other brain regions finding that changes in COMT mRNA levels do not appear to be a predominant mechanism associated with the pathophysiology of schizophrenia.67In addition to changes in dopamine synthesis, transporter and receptor mRNAs, we found changes in mRNA for one catabolic enzyme, MAOA, that can also terminate dopamine action. If there is an increase in dopamine in the synaptic cleft in people with schizophrenia, we suggest that it is possible that the increase in MAOA mRNA may reflect a compensatory response to balance extracellular dopamine levels. However, MAOA is also involved in the breakdown of other neurotransmitters including serotonin and norepinephrine, and MAOA variants are implicated in psychiatric disorders.68 Our gene expression findings are in contrast to a previous post-mortem study showing increased DRD2 receptor binding in midbrain homogenates from drug-naive and drug-treated schizophrenia cases compared with controls;31 thus, we speculate that the changes we find in DRD2 mRNA may result in less DRD2 protein in the presynaptic terminal rather than in the midbrain. The binding of dopamine to the DRD2S autoreceptor on the presynaptic terminal results in the inhibition of dopamine synthesis and release and, thus, a decrease in dopamine neurotransmission.25 We speculate that if the decrease in DRD2S gene expression is reflected in less DRD2S protein expression in the terminal field of the striatum in schizophrenia, this could contribute to an increase in striatal dopamine neurotransmission. In contrast to our evidence of decreased DRD2, we and others, find increased DRD2S mRNA in DLPFC in schizophrenia,69 indicating potential region-specific alterations of this receptor splice variant mRNA in schizophrenia. Although DRD2L and DRD2longer are traditionally thought of as postsynaptic receptors found at terminal sites, we have readily measured mRNA of both of these isoforms in the human midbrain and report decreases in these isoforms in schizophrenia. This suggests that these potential decreases in DRD2 isoforms (short and long) may contribute to presynaptic or dendritic pathophysiology in schizophrenia. Although we did not identify a diagnostic difference in DRD3 or DRD3nf mRNA, schizophrenia cases exhibiting more positive symptoms had a 56% increase in DRD3 mRNA compared with those with more negative symptoms. Thus, increased DRD3 mRNA and possibly protein may also contribute to dopamine dysregulation in some people with schizophrenia.We find decreased DRD2S mRNA in the substania nigra in schizophrenia in our cohort and DRD2S mRNA levels do not correlate with antipsychotic measurements. This supports previous studies showing that treatment with antipsychotics did not change DRD2 mRNA in rodent cortex or striatum.39 However, these studies provide a basis for interpreting changes seen in in vivo studies in schizophrenia patients and the putative clues they provide to pathophysiology, and potential therapeutic targets beyond DRD2 antagonism can be followed up in preclinical models of dopamine dysregulation.In sum, we find evidence of changes in gene expression of multiple molecules that may act individually or in combination to contribute to the dopaminergic system dysregulation identified in people with schizophrenia (see We conclude that, in a demographically well-matched and characterised post-mortem midbrain cohort, gene expression of multiple dopamine-regulating molecules are changed in the substantia nigra in schizophrenia and that changes in autoreception, transport and possible catabolism together may contribute to the dopamine dysregulation in schizophrenia . This st


CIP2A also supports male germ cell proliferation in vivo. However, the role of MYC in normal germ cell proliferation and spermatogonial progenitor self-renewal is currently unclear. Here, we demonstrate that male germ cells are CIP2A-positive but lack detectable levels of MYC protein; whereas MYC is highly expressed in Leydig cells and peritubular myoid cells contributing thereby to the testicular stem cell niche. On the other hand, MYC was co-expressed with CIP2A in testicular cancers. These results demonstrate that CIP2A and MYC are spatially uncoupled in the regulation of spermatogenesis, but functional relationship between these two human oncoproteins is established during testicular cancer transformation. We propose that further analysis of mechanisms of MYC silencing in spermatogonial progenitors may reveal novel fundamental information relevant to understanding of MYC expression in cancer.High MYC expression is linked to proliferative activity in most normal tissues and in cancer. MYC also supports self-renewal and proliferation of many types of tissue progenitor cells. Cancerous inhibitor of PP2A (CIP2A) promotes MYC phosphorylation and activity during intestinal crypt regeneration  Animal reproduction is entirely dependent on adult germ cell proliferation. A special mode of proliferation, the self-renewal of tissue-specific stem - and progenitor cells is required both to support the renewal of tissues and for the rapid production of sperm. Spermatogonia, that reside in the outer basal layer of the seminiferous tubules, are among the most actively dividing cells in human body and therefore characterizing regulators of their self-renewal could reveal generally important insights into the biology of progenitor and stem cells. Moreover, comparative analysis of molecular mechanisms driving testicular proliferation and self-renewal involved in normal spermatogenesis but also in the context of testicular cancers could reveal causal mechanisms generally relevant for cellular transformation.). A PP2A-interacting protein designated cancerous inhibitor of PP2A (CIP2A) inhibits degradation of serine 62 phosphorylated form of the oncoprotein MYC (Myant 2015). Physiologically, CIP2A promotes spermatogenesis by supporting maintenance of spermatogonial progenitor pool (Ventel _2012), and facilitates intestinal crypt progenitor-driven intestinal regeneration in vivo (Myant 2015[Protein phosphatase 2A (PP2A) accounts for a large fraction of the total cellular serine/threonine phosphatase activity , (b) the established functional link between CIP2A and MYC (Myant 2015), and (c) the role of both CIP2A and MYC in regulating progenitor cell fate (Ventel _2012[), we assessed the status of MYC in spermatogonial progenitor cells, spermatogonia. Systemic inhibition of MYC induced dramatic testicular atrophy marked by a loss of spermatogonia and spermatocytes (Soucek 2008). However, to our knowledge MYC protein expression has not been demonstrated from testicular tissue as yet, and thus it has been unclear which testicular cell types express MYC. This is especially the case in the light of earlier results that mouse germ cells appear to proliferate in the absence of MYC transcription (Stewart 1984). Therefore we analysed human testis samples by immunohistochemistry. While the testicular interstitial tissue was strongly immunopositive for MYC, its expression was not detected in the germ cells which were CIP2A positive (Based on (a) expression and functional role of CIP2A in spermatogonia (Ventel _2012tel _2012) as well as proliferation (ki-67) was reduced in the cryptorchid testis as compared to the house-keeping gene L19 . Therefore, to clarify MYC expression pattern in the mouse testis, we performed RT-PCR analyses using three different primers to amplify MYC mRNAs in the whole testis samples and dissected seminiferous tubules . Murine gene L19 . Howevergene L19 . Similargene L19 . These r) and during intestinal regeneration in vivo  suggesting that spermatogonial proliferation is another cellular system in which MYCN can compensate for lack of MYC expression. Interestingly, recent studies have shown that in addition to MYC, CIP2A inhibits PP2A activity towards AKT kinase (Chen 2010). Taking into account the reported role for AKT in promoting self-renewal of isolated spermatogonial stem and progenitor cells (Lee 2007), CIP2A may also exert its effects on spermatogenesis via regulating AKT activity. In addition to the relevance of the present results for understanding CIP2A’s role in spermatogenesis, these findings reveal novel insights into MYC biology. Our results that are based on carefully controlled IHC protocol demonstrate that MYC is not expressed in highly proliferating male germ cells with self-renewal capacity. These results agree with those of mRNA analysis of MYC expression in mouse germ cells (Stewart 1984[). Therefore, our results also suggest a potential role for MYC in the formation of the testicular stem cell niche.Our results strongly indicate that CIP2A promotes spermatogenesis by a mechanism different from its established role of stabilizing MYC in cancer cells. Also, our results are interesting in the light of previous findings that another MYC family gene, MYCN, was shown to be expressed in isolated spermatogonial cells wart 1984). Our reThese results demonstrate that CIP2A and MYC are spatially uncoupled in the regulation of spermatogenesis, but functional relationship between these two human oncoproteins is established during transformation to testicular cancer.) and by three different approaches to demonstrate a lack of correlation between MYC mRNA expression and markers of self-renewal and proliferation in spermatogonia , MYC expression is silenced in normal SPCs but de-repressed in testicular cancer cells. Identifying the molecular mechanisms by which MYC expression is repressed during normal spermatogenesis may lend novel insights into MYC regulation in various physiological and pathological situations. On the other hand, we speculate that germ cell tumorigenesis may arise from the loss of germ line-specific inhibitors of MYC gene expression that in normal germ cells prevent excessive pluripotency and self-renewal, but when absent in abnormal germ cells, result in the conversion to germ cell cancer.Lack of detectable MYC expression in spermatogonial progenitors is an intriguing finding because MYC is prototypically expressed in highly proliferative normal and malignant cells. Based on our and published data  incubation was done for 30 min. Slides, again rinsed, were then incubated in DAB+ liquid Dako (K3468) for 10 min, and then rinsed in water. Samples were incubated in Mayers HTX for 1 min, rinsed with tap water, and finally dehydrated, cleared and mounted. Human testicular samples were obtained from patient diagnosed testicular neoplasm and therefore underwent orchiectomy.Liquid nitrogen frozen mouse samples were homogenized using the MagNA Lyser and MagNA Lyser Green Beads. Briefly, RNA and protein samples were homogeneized in respective lysing buffers, RA1 from Macherey Nagel for RNA; RIPA buffer  for protein samples. 1 to 4 cycles  were used to homogeneize the tissues, an ice-cooling step of 2 min being done between each cycle. Total RNA was extracted and cleaned up from the lysate using the Nucleospin kit (Macherey Nagel), including a DNAse treatment step.), MYC: mouse monoclonal 26 9E10  and S62-MYC: rabbit polyclonal anti-S62-MYC (Escamilla Powers_2006).The following antibodies were used for immunohistochemical stainings of paraffin embedded tissues: CIP2A: rabbit polyclonal anti-CIP2A  were added to 8 μL of a PCR mixture made up of 5 μL of PCR Master Mix (Applied Biosystems), 1 μL of each primer at a concentration of 3 μM, and 1 μL of specific probe at a concentration of 2 μM. The thermal cycling conditions comprised an initial step at 50°C for 2 min and a denaturation step at 95°C for 10 min followed by 40 cycles at 95°C for 15 and 60°C for 1 min. All PCRs were carried out using an ABI Prism 7000 Sequence Detection System (Applied Biosystems). The specificity of each primer couple was shown by a dissociation curve analysis. Results are derived from the average of at least two independent experiments. Gene expression was reported relative to housekeeping gene.Surgical unilateral cryptorchidism (Crypt) of the right testes was induced in nine male mice (C57BL/6) at 8 weeks of age. Each mouse was anesthetized, and a lateral incision was made on the abdominal wall above the inguinal canal. The right testis was pulled away from the scrotum into the abdominal cavity. The inguinal canal was closed with nonabsorbable sutures. The mice were sacrificed after 6 months, and both testes were collected for histological and quantitative polymerase chain reaction (PCR) (qPCR) analyses.Busulfan  was dissolved in dimethyl sulfoxide and mixed with an equal volume of sterile distilled water before intraperitoneal injection into 8 weeks old C57BL/6 mice. Mice were treated with 20 or 30 mg/kg busulfan. Control mice were injected with 30 mg/kg dimethyl sulfoxide. Four weeks after injection the mice were sacrificed and testes were collected for histological and quantitative polymerase chain reaction (PCR) (qPCR) analyses.Supplementary Materials