Deafening-induced rapid changes to spine synaptic connectivity in the adult avian vocal basal ganglia.

The basal ganglia have been implicated in auditory-dependent vocal learning and plasticity in human and songbirds, but the underlying neural phenotype remains to be clarified. Here, using confocal imaging and three-dimensional electron microscopy, we investigated striatal structural plasticity in response to hearing loss in Area X, the avian vocal basal ganglia, in adult male zebra finch (Taeniopygia guttata). We observed a rapid elongation of dendritic spines, by approximately 13%, by day 3 after deafening, and a considerable increase in spine synapse density, by approximately 61%, by day 14 after deafening, compared with the controls with an intact cochlea. These findings reveal structural sensitivity of Area X to auditory deprivation and suggest that this striatal plasticity might contribute to deafening-induced changes to learned vocal behavior.

Remodeling of Dendritic Spines in the Avian Vocal Motor Cortex Following Deafening Depends on the Basal Ganglia Circuit.

Deafening elicits a deterioration of learned vocalization, in both humans and songbirds. In songbirds, learned vocal plasticity has been shown to depend on the basal ganglia-cortical circuit, but the underlying cellular basis remains to be clarified. Using confocal imaging and electron microscopy, we examined the effect of deafening on dendritic spines in avian vocal motor cortex, the robust nucleus of the arcopallium (RA), and investigated the role of the basal ganglia circuit in motor cortex plasticity. We found rapid structural changes to RA dendritic spines in response to hearing loss, accompanied by learned song degradation. In particular, the morphological characters of RA spine synaptic contacts between 2 major pathways were altered differently. However, experimental disruption of the basal ganglia circuit, through lesions in song-specialized basal ganglia nucleus Area X, largely prevented both the observed changes to RA dendritic spines and the song deterioration after hearing loss. Our results provide cellular evidence to highlight a key role of the basal ganglia circuit in the motor cortical plasticity that underlies learned vocal plasticity.

Sexual Differences in Cell Loss during the Post-Hatch Development of Song Control Nuclei in the Bengalese Finch.

Birdsongs and the regions of their brain that control song exhibit obvious sexual differences. However, the mechanisms underlying these sexual dimorphisms remain unknown. To address this issue, we first examined apoptotic cells labeled with caspase-3 or TUNEL in Bengalese finch song control nuclei - the robust nucleus of the archopallium (RA), the lateral magnocellular nucleus of the anterior nidopallium (LMAN), the high vocal center (HVC) and Area X from post-hatch day (P) 15 to 120. Next, we investigated the expression dynamics of pro-apoptotic (Bid, Bad and Bax) and anti-apoptotic (Bcl-2 and Bcl-xL) genes in the aforementioned nuclei. Our results revealed that the female RA at P45 exhibited marked cell apoptosis, confirmed by low densities of Bcl-xL and Bcl-2. Both the male and female LMAN exhibited apoptotic peaks at P35 and P45, respectively, and the observed cell loss was more extensive in males. A corresponding sharp decrease in the density of Bcl-2 after P35 was observed in both sexes, and a greater density of Bid was noted at P45 in males. In addition, we observed that RA volume and the total number of BDNF-expressing cells decreased significantly after unilateral lesion of the LMAN or HVC (two areas that innervate the RA) and that greater numbers of RA-projecting cells were immunoreactive for BDNF in the LMAN than in the HVC. We reasoned that a decrease in the amount of BDNF transported via HVC afferent fibers might result in an increase in cell apoptosis in the female RA. Our data indicate that cell apoptosis resulting from different pro- and anti-apoptotic agents is involved in generating the differences between male and female song control nuclei.

Sexual differences in cell proliferation in the ventricular zone, cell migration and differentiation in the HVC of juvenile Bengalese finch.

Song control nuclei have distinct sexual differences and thus are an ideal model to address how brain areas are sexually differentiated. Through a combination of histological analysis and electrical lesions, we first identified the ventricle site for HVC progenitor cells. We then found that there were significant sex differences in the cellular proliferation activity in the ventricular zone of the HVC, the number of migrating cells along the radial cells (positive immunoreactions to vimentin) and differentiation towards neurons. Through co-culturing of male and female slices containing the developing HVC in the same well, we found that the male slices could produce diffusible substances to masculinize the female HVC. By adding estrogen, an estrogen antagonist, brain-derived neurotrophic factor (BDNF) or its antibody into the culture medium, separately or in combination, we found that these diffusible substances may include estrogen and BDNF. Finally, we found that 1) estrogen-induced BDNF upregulation could be detected 48 hr after estrogen treatment and could not be blocked by a vascular endothelial growth factor (VEGF) receptor inhibitor and 2) the amount of VEGF mRNA expressed in the developing HVC and its adjacent area did not display any significant sex differences, as did the distribution of VEGF and laminin-expressing endothelial cells in the developing HVC. Because these findings are largely different from previous reports on the adult female HVC, it is suggested that our estrogen-induced BDNF up-regulation and the resultant sexual differentiation might not be mediated by VEGF and endothelial cells, but instead, may result from the direct effects of estrogen on BDNF.

Alteration of CaBP expression pattern in the nucleus magnocellularis following unilateral cochlear ablation in adult zebra finches.

Songbirds have the rare ability of auditory-vocal learning and maintenance. Up to now, the organization and function of the nucleus magnocellularis (NM), the first relay of the avian ascending auditory pathway is largely based on studies in non-vocal learning species, such as chickens and owls. To investigate whether NM exhibits different histochemical properties associated with auditory processing in songbirds, we examined the expression patterns of three calcium-binding proteins (CaBPs), including calretinin (CR), parvalbumin (PV) and calbindin-D28k (CB), and their relations to auditory inputs in NM in adult zebra finches. We found enriched and co-localized immunostaining of CR, PV and CB in the majority of NM neurons, without neuronal population preference. Furthermore, they were sensitive to adult deafferentation with differential plasticity patterns. After unilateral cochlear removal, CR staining in the ipsilateral NM decreased appreciably at 3 days after surgery, and continued to decline thereafter. PV staining showed down-regulation first at 3 days, but subsequently recovered slightly. CB staining did not significantly decrease until 7 days after surgery. Our findings suggest that the three CaBPs might play distinct roles in association with auditory processing in zebra finches. These results are in contrast to the findings in the NM of chickens where CR is the predominant CaBP and deafferentation had no apparent effect on its expression. Further extended studies in other avian species are required to establish whether the difference in CaBP patterns in NM is functionally related to the different auditory-vocal behaviors.

Ultrastructural and electrophysiological analysis of Area X in the untutored and deafened Bengalese finch in relation to normally reared birds.

Birdsong learning bears many similarities to human speech acquisition. Although the anterior forebrain pathway (AFP) is believed to be involved in birdsong learning, the underlying neural mechanisms are unclear. We produced two types of abnormal song learning: young birds untutored from adult "song tutors", or birds deafened by bilateral cochlear removal before the onset of sensory learning. We then studied how ultrastructure and electrophysiological activity changed in an AFP nucleus, Area X, among these birds at adulthood. Our results showed that, although the size of Area X did not change significantly, the numbers of synapses per unit area and compound synapses and the percent of concave synapses increased significantly in the untutored or deafened birds. The percent of perforated synapses or axo-spinous synapses decreased compared to the normally reared birds, suggesting a decreased efficiency of synaptic transmission in the untutored or deafened birds. We then identified several types of spontaneously firing cells in Area X. Cells with fast and slow firing rates did not show significant electrophysiological differences among the groups, but cells with moderate firing rates, most likely DLM-projecting neurons, fired at significantly lower rates in the untutored and deafened birds. In addition, cells firing irregularly were only found in the deafened birds. Thus, the decreased or irregular electrophysiological activity in the untutored or deafened birds, together with the corresponding ultrastructural findings, could be implicated in the abnormal song production in these two types of birds.

Living high training low induces physiological cardiac hypertrophy accompanied by down-regulation and redistribution of the renin-angiotensin system.

AIM: Living high training low" (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O2. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats. METHODS: Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT1) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively. RESULTS: LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT1 genes, but increased the expression of ACE and AT1 proteins in heart tissue. Moreover, ACE and AT1 protein expression was significantly increased in the endocardium, but unchanged in the epicardium. CONCLUSION: LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT1 protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.

Changes in ultra-structures and electrophysiological properties in HVC of untutored and deafened Bengalese finches relation to normally reared birds: implications for song learning.

Songbirds are increasingly used as an experimentally tractable system to study the neurobiological underpinnings of vocal learning. To gain additional insights into how birdsongs are learned, we compared the size of HVC, the high vocal center for song production, and its ultrastructural or electrophysiological properties between the normally reared Bengalese finches, and the untutored or deafened ones before the onset of sensory learning (around post-hatching day 20). Our results showed that HVC had more synapses and concave synaptic curvature, but fewer perforated synapse, in the untutored or deafened birds in comparison with those in the normally reared birds. Although there was no significant difference of the ratio of straight or compound synapses, there was an increasing tendency for the untutored and deafened birds to possess more straight and compound synapses. These data revealed that synapses in the isolated or deafened birds had lower synapse activity in relation to those with normal hearing. This was confirmed by our electrophysiological results to show significant decreases in the firing rates of spike or burst in the isolated or deafened birds in the three types of HVC neurons i.e., putative X-projecting neurons, RA-projecting neurons and interneurons. In addition, low firing frequency (<10Hz) occurred much more in the above three types of HVC neurons in the tutored or deafened birds than in the normally reared birds. These data suggest that all the three putative types of neurons in HVC might be involved in the activity of the production of adult normal songs.

Comparative study on song behavior, and ultra-structural, electrophysiological and immunoreactive properties in RA among deafened, untutored and normal-hearing Bengalese finches.

To gain additional insight into how a birdsong is learned, we compared the songs of Bengalese finch males that were deafened early in development or raised without tutors to control finches that learned songs from adult models. Fewer note types and a more variable number of notes per bout were observed in untutored male songs, and no audible songs were detected in deafened males. We then investigated the ultrastructural, immunohistological, and electrophysiological correlates of the outcomes of song learning within the robust nucleus of the archopallium (RA), a forebrain nucleus for song production. In comparison to control birds, untutored and deafened birds had more synapses per unit volume, fewer vesicles per synapse, longer postsynaptic densities, and a lower proportion of perforated synapses, which suggest lower activity or decreased efficiency of synaptic transmission within the RA of the treated birds. For anesthetized birds, neurons within the RA of untutored and deafened males had lower spontaneous firing rates, fewer and shorter bursts, and higher coefficient of variation of the instantaneous firing rate than the normally reared males. Compared with controls, the untutored and deafened males had higher staining intensities within the RA of GABA and the GABA(A) receptor, less staining of tyrosine hydroxylase and no difference in the staining of NMDA receptors. Thus, both the ultrastructural and immunohistochemical results could explain for the stronger electrophysiological activities in normally reared birds. Because RA is involved in generating the motor commands, these data might account for the deficits in birds with abnormal song learning.

Sites of origin and developmental dynamics of the neurons in the core and shell regions of torus semicircularis in the Chinese softshell turtle (Pelodiscus sinensis).

To know the embryogenesis of the core and shell regions of the midbrain auditory nucleus, a single dose of [(3)H]-thymidine was injected into the turtle embryos at peak stages of neurogenesis in the shell and core of the torus semicircularis. Following sequential survival times, labeled neurons and the dynamics of cell proliferation were examined. The expression of vimentin (VM), reelin, calbindin, parvalbumin, and substance P were also studied. The results showed that: 1) progenitor cells for the core and shell regions were generated in different sites of the ventricular zone; 2) the length of the cell cycle or S-phase for the shell region were both longer than those for the core region (4.7 and 3.2 hours longer, respectively), suggesting that mitotic activity in the core region is higher than it is in the shell region; 3) the elongated cell bodies of the labeled core and shell cells had close apposition to VM fibers, suggesting that the migration of these cells is guided by VM fibers; 4) the germinal sites of the core and shell constructed by projecting the orientation of radial VM fibers back to the ventricular zone was consistent with those obtained by short and sequential survival [(3)H]-thymidine radiography; and 5) the beginning of positive staining for parvalbumin in the core region was interposed between those for calbindin and substance P in the shell regions. This study contributes to the understanding of how auditory nuclei are organized and how their components developed and evolved.

Comparative study on the song behavior and song control nuclei in male and female Mongolian larks (Melanocorypha mongolica).

Songbirds can produce a remarkable diversity of songs, which is well-characterized learned behavior that reflects the basic processes of language learning in humans. As song control nuclei governing song behavior has been identified, bird song provides an excellent model to address the relationship between brain areas and their controlling behavior. The Mongolian lark (Melanocorypha mongolica), a species of the Alaudidae family, is well known for its elaborate singing and ability to learn new songs, even in adulthood. Here, we studied the singing behavior and underlying neural structures of the Mongolian lark in both sexes. We found that the sizes of song bouts and song phrases (song repertoires) in male Mongolian larks are extremely large, and that each song repertoire or phrase has a complex structure, comprising several different syllables that seldom appear in other types of song bouts. In accordance with these complex songs, Mongolian lark song control nuclei are well developed and can be easily detected by Nissl staining. In contrast to male Mongolian larks, females were not observed to sing. However, they possess significant song control nuclei with abundant neural connectivity within them despite their small sizes compared with males. These data provide new evidence that help further clarify the mechanisms by which songbirds sing. Our results also have implications for the evolution of complex birdsongs and song control nuclei in oscine birds.

Germinal sites and migrating routes of cells in the mesencephalic and diencephalic auditory areas in the African clawed frog (Xenopus laevis).

There is a clear core-shell organization in the auditory nuclei of amniotes. However, such organization only exists in the mesencephalic, but not in the diencephalic auditory regions of amphibians. To gain insights into how this core-shell organization developed and evolved, we injected a small dose of [(3)H]-thymidine into tadpoles of Xenopus laevis at peak stages of neurogenesis in the mesencephalic and diencephalic auditory areas. Following different survival times, the germinal sites and migrating routes of cells were examined in the shell (laminar nucleus, Tl; magnocellular nucleus, Tmc) and core (principal nucleus, Tp) regions of the mesencephalic auditory nucleus, torus semicircularis (Ts), as well as in the diencephalic auditory areas (posterior thalamic nucleus, P; central thalamic nucleus, C). Double labeling for [(3)H]-thymidine autoradiography and immunohistochemistry for vimentin was also performed to help determine the routes of cell migration. We found three major results. First, the germinal sites of Tp were intercalated between Tl and Tmc, arising from those of the shell regions. Second, although the germinal sites of Tl, Tmc, and Tp were located in the same brain levels (at rostromedial or caudomedial levels of Ts), neurogenesis in Tl or Tmc started earlier than that in Tp. Finally, the P and C were also generated in different ventricle sites. However, unlike Ts their neurogenesis showed no obvious temporal differences. These data demonstrate that a highly differentiated auditory region, such as Tp in Ts, is lacking in the diencephalon of amphibian. Our data are discussed from the view of the constitution and evolutionary origins of auditory nuclei in vertebrates.

Identification of male-biased gene: parvalbumin in song control nuclei of the Bengalese finch.

Sex differences in song nuclei are evident across songbirds. To explore candidate genes involving in the sexual dimorphism of song nuclei, the present study used suppression subtraction hybridization to identify male-biased genes in the Bengalese finch (Lonchura striata). From 199 clones with an inserted sequence, we obtained a gene (parvalbumin, PV) coding a calcium-binding protein, which showed, through semi-quantitative PCR, obviously male-biased expression. In situ hybridization and immunohistochemistry indicated that PV was sexually distributed in most of the studied song nuclei, including in the high vocal center (HVC), the robust nucleus of the arcopallium (RA), Area X, and the lateral magnocellular nucleus of the anterior nidopallium (lMAN) for three studied age groups, namely, posthatching day (PD) 15, 45, and adult. The total number of PV mRNA or protein cells was significantly larger in males in the HVC, RA, and Area X for PD45 and adult. Considering that calcium-binding proteins have reported effects on the maturation of some brain areas, and on the sexual differentiation of mammalian brain areas by affecting cell survival rates, our study suggests that PV may be involved in the functional maturation of neurons in song nuclei or the sexual differentiation of song system.

Reversal of MDR1 gene-dependent multidrug resistance in HL60/HT9 cells using short hairpin RNA expression vectors.

Multidrug resistance (MDR) is a serious obstacle to cancer chemotherapy. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers MDR to tumor cells. This study explored the possibility of reducing drug resistance by targeting the mdr1 gene using short hairpin RNA (shRNA). Two different shRNAs were designed and constructed in a pSilencer 2.1-U6 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. Real-time polymerase chain reaction and Western blotting were used to characterize the inhibited expression of MDR1 mRNA and P-gp, and the drug sensitivity of the transfected cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The results indicated that the inhibition of P-gp expression by small interfering RNA selectively restored sensitivity to the drugs transported by P-gp. Evaluation of chemosensitivity showed 52.58% reversal by p2.1-shRNA1 and 73.07% reversal by p2.1-shRNA2 in drug resistance for harringtonine, and 84.87% reversal by p2.1-shRNA1 and 94.23% reversal by p2.1-shRNA2 in drug resistance for curcumin in the transfected cells. The results demonstrated the efficacy and selectivity of shRNA in reversing MDR in drug-resistant HT9 cells in vitro.

Song control nuclei in male and female large-billed crows (Corvus macrorhynchos).

We show that the learned vocalizations of male and female large-billed crows (Corvus macrorhynchos) are similar and that their functions and physical features show significant differences from those of other oscine species. We investigate whether the song control nuclei of crows show any sexual differences in size, reflecting differences in their singing behavior, and whether these nuclei are different from those of other songbirds in terms of neural connectivity size and relative to the forebrain. Our Nissl staining results reveal that 1) of the four song nuclei examined (HVC; the robust nucleus of the arcopallium [RA]; Area X; and the dorsolateral medial nucleus [DLM]), HVC, RA, and Area X volumes are significantly larger in males than in females, but DLM volume and body and brain weights show no significant gender differences; and 2) the sizes of song nuclei relative to the forebrain are within the range of other oscines. By injecting a neural tract tracer (DiI) into various song nuclei in brain slices, we found that, as in other songbirds, HVC projects to RA and Area X, while Area X projects to the lateral magnocellular nucleus of the anterior nidopallium (IMAN) and DLM, DLM to IMAN, and IMAN to RA. Our results Indicate that, although the crow has songs very different from those of other oscine species, Its song nuclei and the connections between them are not obviously different.

Evolutionary significance of delayed neurogenesis in the core versus shell auditory areas of Mus musculus.

Early comparative embryogenesis can reflect the organization and evolutionary origins of brain areas. Neurogenesis in the auditory areas of sauropsids displays a clear core-to-shell distinction, but it remains unclear in mammals. To address this issue, [3H]-thymidine was injected into pregnant mice on consecutive embryonic (E) days (E10-E19) to date neuronal birthdays. Immunohistochemistry for substance P, calbindin, and parvalbumin was conducted to distinguish the core and shell auditory regions. The results showed that: 1) cell generation began at E13 in the external or dorsal nucleus of the inferior colliculus (IC), but it did not start in the caudomedial portion of the central nucleus of IC, and significantly fewer cells were produced in the medial and rostromedial portions of the central nucleus of IC; 2) cells were generated at E11 in the dorsal and medial divisions of the medial geniculate complex (MGd and MGm, respectively), whereas cell generation was absent in the medial and rostromedial portions of the ventral medial geniculate complex (MGv), and fewer cells were produced in the caudomedial portion of MGv; 3) in the telencephalic auditory cortex, cells were produced at E11 or E12 in layer I and the subplate, which receive projections from the MGd and MGm. However, cell generation occurred at E13-E18 in layers II-VI, including the area receiving projections from the MGv. The core-to-shell distinction of neurogenesis is thus present in the mesencephalic to telencephalic auditory areas in the mouse. This distinction of neurogenesis is discussed from an evolutionary perspective.

Collapsin response mediator protein-4 (CRMP-4) expression in posthaching development of song control nuclei in Bengalese finches.

CRMP-4 is regarded to play a role in neuronal differentiation, neurite growth and synapse formation. It has been shown to express in brain areas undergoing plastic changes or neuronal generation. Bird song is a learned, complex behavior. During song learning, some neural changes occur dramatically within song nuclei in neuron number, neuronal morphology, and synaptic formation or rearrangements. In order to get insights into the potential functions of CRMP-4 in the posthatching development of song nuclei during song learning, we examined the expression of CRMP-4 protein and mRNA in song control nuclei of Bengalese finch (Lonchura striata) from posthatching days (P) 10 to adulthood. Our study showed that cells positive for CRMP-4 protein and mRNA were distributed in song nuclei nearly in all the studied groups. The numbers of CRMP-4 cells in most of studied song nuclei changed significantly with age. They reached the peak at P15 in the lateral magnocellular nucleus of anterior nidopallium (LMAN) and the caudal medial nidopallium (NCM), or at P25 in HVC, Area X and the dorsolateral nucleus of the medial anterior thalamus (DLM). They then continued to decrease till adulthood. CRMP-4 protein and mRNA were both relatively high expressed during the post-hatch development of song control nuclei and song learning (P20-60), suggesting that CRMP-4 is involved in these activities. Although CRMP-4 protein and mRNA largely decreased at adulthood, they continued to express moderately, revealing that CRMP-4 may play a role in the maintenance of adult song nuclei.

Comparative analysis of neurogenesis between the core and shell regions of auditory areas in the chick (Gallus gallus domesticus).

Early embryogenesis can reflect constituting organizations and evolutionary origins of brain areas. To determine whether a clear core-versus-shell distinction of neurogenesis that occurs from the auditory midbrain to the telencephalon in the reptile also appears in the bird, a single dose of [(3)H]-thymidine was injected into chick (Gallus gallus domesticus) eggs at some successive embryonic days (E) (from E3 to E10). Towards the end of hatching, [(3)H]-thymidine labeling was examined, and the results were as follows: 1) Neuronal generation in the nucleus intercollicularis (ICo) (shell region) began at E3, whereas neurogenesis began at E4 in the nucleus mesencephalicus lateralis pars dorsalis (MLd) (core region); 2) Neurogenesis initiated at E3 in the nucleus ovoidalis (Ov) shell, but initiated at E4 in the rostral Ov core. In the medial or caudal Ov core, the percentage of heavily-labeled neurons with [(3)H]-thymidine was significantly lower at E3 age group than that in the Ov shell; 3) In field L1 and L3, two flanking regions of the primary telencephalic auditory area (field L2a), neurogenesis started at E5, but started at E6 in field L2a. These data indicate that the onset of embryogenesis began earlier in the auditory shell areas than in the core areas from the midbrain to the telencephalon. These findings provide insight into the organization of auditory nuclei and their evolution in amniotes.

The reversion effect of the RNAi-silencing mdr1 gene on multidrug resistance of the leukemia cell HT9.

Overexpression of P-glycoprotein (P-gp), the mdr1 gene product, confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. This study evaluated RNAi for specific silencing of the mdr1 gene and reversion of multidrug resistance. Three different short hairpin RNAs (shRNAs) were designed and constructed in a pSilencer 3.1-H1 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. The RNAi effect was evaluated by real-time PCR, Western blotting and cell cytotoxicity assay. In the cell, shRNAs can specifically down-regulate the expression of mdr1, mRNA and P-gp. Resistance against harringtonine, doxorubicin and curcumin was decreased. The study indicated that shRNA recombinant plasmids could modulate MDR in vitro.