Chronic morphine increases D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA, but has no influence on those of projection neurons from the hippocampus or the thalamus to the BLA
To study the effect of chronic morphine treatment on the expression of dopamine D1 receptor (D1R) in glutamatergic terminals of projection neurons from the mPFC to the BLA, we observed the influence of chronic morphine on the coexpression of D1R, vesicular glutamate transporters 2 (VGLUT2), and biotinylated dextran amine (BDA) in slices of the BLA. Here, we used BDA as an anterograde tracer because it was preferentially transported anterogradely and could label axons and terminals [21]. The top and bottom panels of Fig. 1a show D1R staining (column 1, green color, Fig. 1a), BDA labeling (column 2, red color, Fig. 1a), glutamatergic terminal marker VGLUT2 immunoreactivity (column 3, blue color, Fig. 1a), and coexpression of D1R, BDA, and VGLUT2 (column 4, white color, Fig. 1a) in slices of the BLA in the saline and chronic morphine groups. Figure 1b showed the fluorescence intensity of green, red, blue, and white color, which was quantified using MacBiophotonics Image J software in a series of stained sections from chronic morphine- or saline-treated animals. The results showed that chronic morphine did not change the fluorescence density of BDA (panel 2, Fig. 1b) and VGLUT2 (panel 2, Fig. 1b), but significantly increased the fluorescence density of D1 receptors (panel 1, Fig. 1b, saline group: 100.0 ± 13.5%, n = 6; morphine group: 140.5 ± 10.9%, n = 6, independent t test, P = 0.009, compared to saline group) and the coexpression of D1R, BDA, and VGLUT2 in the BLA (panel 3, Fig. 1b, saline group: 100.0 ± 14.3%, n = 6; morphine group: 158.6 ± 9.8%, n = 6, independent t test, P = 0.007, compared to saline group). This result suggested that chronic morphine could significantly increase the expression of D1 receptors in the BLA and in glutamatergic terminals of projection neurons from the mPFC to the BLA.
We also examined the influence of chronic morphine on the expression of D1 receptors in glutamatergic terminals of projection neurons from the hippocampus or the thalamus to the BLA using a similar method. Figure 2a shows the fluorescence intensity of green, red, blue, and white color quantified using MacBiophotonics Image J software in stained glutamatergic terminals of projection neurons from the hippocampus to the BLA sections from the saline and chronic morphine groups. The results indicate that chronic morphine did not change the fluorescence density of BDA (panel 2, Fig. 2a), VGLUT2 (panel 2, Fig. 2a), and the coexpression of D1R, BDA, and VGLUT2 in the BLA (panel 3, Fig. 2a, saline group: 100.0 ± 7.3%, n = 9; morphine group: 111.3 ± 3.3%, n = 9, independent t test, P = 0.175, compared to saline group), but significantly increased the fluorescence density of D1 receptors (panel 1, Fig. 2a, saline group: 100.0 ± 10.3%, n = 9; morphine group: 134.4 ± 13.3%, n = 9, independent t test, P = 0.013, compared to saline group). These results suggest that chronic morphine has no significant influence on the expression of D1 receptors in glutamatergic terminals of projection neurons from the hippocampus to the BLA, although it has an influence on the whole expression of D1 receptors in the BLA. In addition, Fig. 2b shows the fluorescence intensity of green, red, blue, and white color, which was quantified using MacBiophotonics Image J software in stained glutamatergic terminals of projection neurons from the thalamus to the BLA sections from the saline and chronic morphine groups. The results indicate that chronic morphine did not change the fluorescence density of BDA (panel 2, Fig. 2b), VGLUT2 (panel 2, Fig. 2b), and the coexpression of D1R, BDA, and VGLUT2 in the BLA (panel 3, Fig. 2b, saline group: 100 ± 5.5%, n = 6; morphine group: 105.5 ± 4.6%, n = 6, independent t test, P = 0.157, compared to saline group), but significantly increased the fluorescence density of D1 receptors (panel 1, Fig. 2b, saline group: 100.0 ± 9.8%, n = 6; morphine group: 139.3 ± 12.4%, n = 6, independent t test, P = 0.018, compared to saline group). These results suggest that chronic morphine had no significant influence on the expression of D1 receptors in glutamatergic terminals of projection neurons from the thalamus to the BLA, although it had an influence on the whole expression of D1 receptors in the BLA.
There are two kinds of drug administration paradigm in an animal addiction study, wherein one is a non-contingent (e.g., experimenter-administered) paradigm and the other is a contingent (self-administered drug) paradigm. Since human addiction involves drug self-administration, the validity of the latter model exceeds that of the former. However, the non-contingent model is still used because of the relative ease of drug delivery and the overlap in some of the biological changes elicited by contingent and non-contingent drug administration [22]. To examine whether contingent and non-contingent morphine exposure have a similar effect on D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA, we observed the effect of self-administration of morphine on D1 receptor expression in glutamate terminals of projection neurons from the mPFC to the BLA using the self-administration paradigm. The self-infusion number during the period of morphine self-administration is presented in Fig. 3a. Two-way ANOVA for repeated measures revealed that the self-infusion number had a treatment × time interaction effect (two-way ANOVA, F
(6, 60) = 47.66, P < 0.001). There was a significant difference between the number of self-infusions in the saline and morphine groups on day 7 (Bonferroni post hoc analysis, t = 12.75, P < 0.001). At 4 h after administration of the last test, D1R staining, BDA labeling, glutamatergic terminal marker VGLUT2 immunoreactivity, and coexpression of D1R, BDA and VGLUT2 were examined. The results showed that, similar to the experimenter-administered paradigm, self-administration of morphine also significantly increased the expression of D1 receptors in the BLA (saline self-administration group: 100 ± 13.8%, n = 6; morphine self-administration group: 145.8 ± 15.7%, n = 6, independent t test, P = 0.009, compared to saline self-administration group) and in glutamatergic terminals of projection neurons from the mPFC to the BLA (Fig. 3b, saline self-administration group: 100 ± 21.3%, n = 6; morphine self-administration group: 170.0 ± 19.6%, n = 6, independent t test, P = 0.002, compared to saline self-administration group).
To determine the potential relevance of chronic morphine-induced increases in the expression of D1 receptors in glutamatergic terminals of projection neurons from the mPFC to the BLA to the retrieval of opiate withdrawal memory, we examined whether the expression of D1 receptors in these glutamatergic terminals remained at a high level before measuring CPA in morphine-withdrawal rats. The results showed that D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA remained at a high level prior to CPA measurement in morphine-withdrawal rats (saline group before measuring CPA: 100 ± 16.7%, n = 6; chronic morphine group before measuring CPA: 147.2 ± 28.9%, n = 6, independent t test, P = 0.006, compared to saline group). This result suggests that the high expression of D1 receptors in glutamatergic terminals of projection neurons from the mPFC to the BLA may play a role in CPA.
Chronic morphine inhibits the expression of miR-105 in the mPFC, which results in enhanced D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA
To study how chronic morphine treatment induced an increase in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA, we firstly examined the effect of chronic morphine treatment on the expression of D1 receptor mRNA levels in projection neurons from the mPFC to the BLA using a retrograde tracing technique combined with a single-cell RT-PCR method. Fluorescent microspheres were injected into the BLA to label neurons from the mPFC to the BLA retrogradely. The cytoplasmic content of the labeled cell was harvested by a patch pipette to detect D1 receptor mRNA at a single cell level using a single-cell RT-PCR kit. The results showed that chronic morphine had no significant influence on D1 receptor mRNA level in mPFC-BLA projection neurons. The average percentage of the expression of D1 receptor mRNA in saline and chronic morphine groups was 100.0 ± 15.9% (n = 6) and 92.3 ± 11.4% (n = 6), respectively (independent t test, P = 0.697, compared to saline group).
To explore whether chronic morphine regulated the expression of D1 receptors at post-transcriptional level, we examined the influence of chronic morphine on microRNA (miRNA) expression profile in the mPFC using miRNA microarray technology (Additional file 1). Figure 4a shows the hierarchical clustering analysis of differentially expressed miRNAs in the mPFC from morphine- and saline-treated rats. In this figure, the green color indicates a decrease in miRNA expression, whereas the red color indicates an increase in miRNA expression as compared to control. We found that 97 miRNAs (59 upregulated and 38 downregulated) were altered more than two-fold in the mPFC from chronic morphine-treated rats. Further bioinformatics analysis using the online prediction tools (Targetscan, http://www.targetscan.org and miRanda, http://www.microRNA.org) showed that miR-105 was the only one to be downregulated and related to the D1 receptor gene (Drd1a) (panel 1, Fig. 4b). The basic principle of predicting the relationship between miR-105 and the D1 receptor using these two tools follows the seed sequence of microRNA (2–7 nt) binding to the 3′-untranslated region (3′UTR) of the target gene through complementary base pairing. Therefore, we further investigated the role of miR-105 in chronic morphine-induced increases in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA.
First, we used real-time RT-PCR to confirm the chronic morphine-induced decrease in expression of miR-105 in the mPFC. The result showed that chronic morphine indeed induced a decrease in the expression of miR-105 in the mPFC (panel 2, Fig. 4b, n = 8, paired t test, P = 0.002, compared to saline group). Interestingly, chronic morphine had no significant influence on the expression of miR-105 in the hippocampus and the thalamus. The average percentage of miR-105 level in the chronic morphine group was 91.0 ± 7.3%, showing no significant difference from that of the saline group (100.0 ± 18.5%) (panel 3, Fig. 4b, n = 4, paired t test, P = 0.743, compared to saline group) in the hippocampus. The average percentage of miR-105 level in chronic morphine group was 122.6 ± 27.7%, showing no significant difference with that of the saline group (100.0 ± 32.5%) (panel 4, Fig. 4b, n = 5, paired t test, P = 0.098, compared to saline group) in the thalamus. Then, we examined whether Drd1 was indeed a target of miR-105. Using Targetscan (http://www.targetscan.org) and miRanda (http://www.microRNA.org) prediction tools, it was predicted that miR-105 might bind to the 578–584 position of the 3′UTR of D1 mRNA via its 2–7 nucleotide seed sequence (upper panel of Fig. 4c). To confirm this binding, we constructed wt and mutant type (mut) 3′UTR plasmids of D1 mRNA and detected whether miR-105 could directly target D1 mRNA using a dual luciferase reporter assay method. The result showed that the miR-105 mimics reduced the luciferase activity of wt 3′UTR of D1 mRNA by 25.6 ± 2.0% (n = 3, paired t test, P = 0.009) in comparison with that of miRNA negative control (NC), but did not alter the luciferase activity of mut 3′UTR of D1 mRNA (n = 3, paired t test, P = 0.75, bottom panel of Fig. 4c). This result suggests that miR-105 can directly target 3′UTR of D1 mRNA.
We further examined the influence of decreased miR-105 on D1 receptor expression. To do this, the miR-105 inhibitor (sequence complementary to miR-105) was transfected into primary cultured neurons of the mPFC. The result showed that miR-105 inhibitor-transfected cells exhibited an increase in D1 receptor expression (Fig. 5a, n = 5, paired t test, P = 0.0019, compared to miR-105 inhibitor NC group). In contrast, when we transfected the miR-105 mimic (same sequence as mature miR-105) to primary cultured neurons of mPFC, the expression of D1 receptors was suppressed (Fig. 5b, n = 5, paired t test, P = 0.03, compared to miR-105 NC group).
To study the role of chronic morphine-induced decrease of miR-105 in the expression of D1 receptors in the glutamatergic terminals of projection neurons from the mPFC to the BLA, we examined the effect of chronic morphine on the expression of miR-105 in projection neurons from the mPFC to the BLA using a retrograde tracing technique combined with single-cell RT-PCR. Fluorescent microspheres were injected into the BLA to label neurons from the mPFC to the BLA retrogradely. The left panel of Fig. 6a shows a labeled neuron in the mPFC. The cytoplasmic content of the labeled cell was harvested by a patch pipette (middle panel of Fig. 6a) to detect miR-105 at a single cell level using single-cell RT-PCR kit. The results showed that the average percentage of miR-105 level in the chronic morphine group was 41.6 ± 7.0%, which was significantly lower than that in the saline group (100.0 ± 24.1%) (right panel, Fig. 6a, n = 5, paired t test, P = 0.049, compared to saline group). This result suggests that chronic morphine induces a decrease in the expression of miR-105 in projection neurons from the mPFC to the BLA. On this basis, we observed the influence of the intra-mPFC injection of miR-105 inhibitor on D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA using a triple immunofluorescence staining method. The result showed that the intra-mPFC injection of miR-105 inhibitor could significantly increase the expression of D1 receptors in the glutamatergic terminals of projection neurons from the mPFC to the BLA (column 4, left panel, Fig. 6b, white color). The average percentage of the normalized D1 receptor intensity in the glutamatergic terminals of projection neurons from the mPFC to the BLA was 100.0 ± 16.2% in miR-105 inhibitor NC-lentiviral vector (LV) group (n = 5) and 234.8 ± 47.5% in miR-105 inhibitor-LV group (n = 5, independent t test, P = 0.028, compared to miR-105 inhibitor NC-LV group, right panel, Fig. 6b).
Chronic morphine-induced increase in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA results in sensitization to the effect of D1 receptor agonist on glutamate release
To study the functional consequence of chronic morphine-induced increases in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA, we used an optogenetic method to selectively stimulate excitatory fibers projecting from the mPFC to the BLA and then examined the effect of D1 receptor agonist SKF38393 on mPFC-to-BLA glutamatergic synaptic transmission under chronic morphine or saline conditions. The AAV-CaMKIIα-ChR2-mCherry virus was stereotaxically delivered into the mPFC; the left panel of Fig. 7a shows the expression of ChR2-mCherry in the mPFC after the injection of the virus. The virus was allowed to express for a minimum of 6 weeks in order to have sufficient opsin accumulation in the axons in the BLA (middle panel of Fig. 7a). Whole-cell recording was made in pyramidal cells of the BLA and optical stimulation of ChR2-mCherry-positive fibers from the mPFC to the BLA produced excitatory postsynaptic currents (EPSCs) in pyramidal cells of the BLA and these currents were blocked by bath application of AMPA receptor antagonist DNQX (10 μM) (right panel, Fig. 7a). This result demonstrated that these currents were AMPA receptor mediated. On this basis, we examined the effect of D1 receptor agonist SKF38393 on light-evoked EPSCs in the saline and chronic morphine groups. The left panel of Fig. 7b showed typical traces recorded from BLA neurons after the optical stimulation of the mPFC-to-BLA fibers before and at 15 min after SKF38393 (10 μM) addition in response to 5 ms light pulse in the saline and chronic morphine groups. From these raw traces, we could see that SKF38393 had no significant effect on the amplitude of light-evoked EPSCs in the saline group, but in the chronic morphine group, SKF38393 could significantly increase the amplitude of light-evoked EPSCs. The time course of EPSC response after SKF38393 in the two groups also showed a similar result (middle panel, Fig. 7b, n = 8, from five animals). The average amplitude of light-evoked EPSCs was 169.8 ± 8.7 pA before and 172.6 ± 11.6 pA at 15 min after SKF38393 addition in the saline group (right panel, Fig. 7b, n = 8, from five animals), but in the chronic morphine group, the average amplitude of light-evoked EPSCs increased from 180.6 ± 18.9 pA before to 234.9 ± 26.1 pA at 15 min after SKF38393 addition (right panel, Fig. 7b, n = 8, from five animals, one-way ANOVA, F
(3, 28) = 3.001, P = 0.047).
To investigate the effect of SKF38393 on presynaptic glutamate release, we observed the influence of SKF38393 on paired-pulse facilitation (PPF) of light-evoked EPSCs in the saline and chronic morphine groups. PPF, measured as the ratio of EPSC amplitude in response to two successive stimulation pulses, is a frequently used parameter to monitor presynaptic glutamate release [23]. The results showed that, in the saline group, SKF38393 had no effect on PPF, but in the chronic morphine group, after the addition of SKF38393, the first EPSC was increased by 31.5 ± 3.3% (n = 5, from three animals), whereas the second synaptic response was decreased by 1.6 ± 4.3% (n = 5, from three animals). Therefore, the superimposition of the two traces normalized to the first EPSC at 15 min after SKF38393 addition in the saline and chronic morphine groups revealed that PPF did not change after SKF38393 addition in the saline group, but decreased after SKF38393 addition in the chronic morphine group (left panel, Fig. 7c). The average PPF was 1.2 ± 0.1 before and 1.2 ± 0.1 at 15 min after SKF38393 addition in the saline group (middle panel, Fig. 7c, n = 5, from three animals, paired t test, P = 0.904, compared to control before SKF38393). However, the average PPF was decreased from 1.3 ± 0.1 to 1.0 ± 0.1 at 15 min after SKF38393 addition in the chronic morphine group (right panel, Fig. 7c; n = 5, from three animals, paired t test, P = 0.0299, compared to control before SKF38393). This result suggests that the D1 receptor agonist SKF38393 has no significant effect on glutamate release from the mPFC-to-BLA fibers in pyramidal cells of the BLA under normal conditions, but after chronic morphine, it can significantly increase it.
We also examined the influence of chronic morphine on the basic parameters of excitatory synaptic transmission between mPFC-to-BLA fibers and pyramidal cells of the BLA. We used PPF as the index of presynaptic glutamate release as described above and the ratio of light-evoked AMPAR-mediated EPSCs to NMDAR-mediated EPSCs (AMPAR/NMDAR ratio) as the index of glutamatergic synaptic strength [24]. The results showed that chronic morphine had no significant influence on the PPF of light-evoked EPSCs, but increased the AMPAR/NMDAR ratio. The average PPF in the chronic morphine group was 1.03 ± 0.05, with no significant difference with that of the saline group (1.10 ± 0.07) (right panel, Fig. 8a; n = 8, independent t test, P = 0.409, compared to saline group). The average AMPAR/NMDAR ratio in the chronic morphine group was 1.02 ± 0.04, which was significantly increased compared to the saline group (0.56 ± 0.03) (right panel, Fig. 8b; n = 8, independent t test, P < 0.001, compared to saline group). These results suggest that chronic morphine may not induce a direct change in glutamate release from the mPFC-to-BLA fibers in pyramidal cells of the BLA, but may cause a change in glutamatergic synaptic strength.
Withdrawal-associated environmental cues can activate mPFC-to-BLA projection neurons in morphine-withdrawal rats
To examine whether withdrawal-associated environmental cues could activate mPFC-to-BLA projection neurons, we used a precipitated withdrawal model. In this conventional model of withdrawal, opiate receptor antagonist naloxone is administered to morphine-dependent animals in order to precipitate withdrawal syndromes and for these to occur more reliably [25]. The procedure for the CPA test was similar to that described previously [26]. The rats were given a pre-test to assess their baseline place preference. Then, the rats were confined to withdrawal-paired compartments based on their baseline place preference in the training phase. The post-test was performed by re-exposure of rats to the chamber to freely explore all three compartments. Rats were divided four groups as saline + saline, saline + naloxone, chronic morphine + saline, and chronic morphine + naloxone. The results showed that the rats in the chronic morphine + naloxone group exhibited a strong aversion to withdrawal-paired compartment and thus spent less time in the withdrawal-paired compartment during the post-test than during the pre-test, producing an increase in ‘aversion score’ (CPA score), whereas rats in other groups did not exhibit a significant aversion to either compartment. The average CPA score in the chronic morphine + naloxone group was −342.1 ± 107.2 s (Fig. 9a,
n = 6, two-way ANOVA, Bonferroni post hoc analysis, F
(3, 40) = 7.268, P = 0.0025), but in the saline + saline, saline + naloxone, and chronic morphine + saline groups, the rats had no significant aversive responses for the two compartments (Fig. 9a,
n = 6). On this basis, we examined the effect of withdrawal-associated environmental cues on the expression of c-Fos in mPFC-to-BLA projection neurons in the saline + saline, saline + naloxone, chronic morphine + saline, and chronic morphine + naloxone groups using a retrograde tracing technique combined with c-Fos detecting method. Fluorogold (FG) was injected into the BLA to label neurons from the mPFC to the BLA retrogradely. The effect of withdrawal-associated environmental cues on the expression of c-Fos in FG-labeled neurons of the mPFC was examined. The upper line in Fig. 9b showed c-Fos expression (red color) and the middle line showed FG-labeled neurons (green color) of the mPFC in these four groups. The bottom line of Fig. 9b showed the co-labeling (yellow color) of c-Fos and FG. The co-labeling of c-Fos and FG significantly increased in the chronic morphine + naloxone group when rats were exposed to withdrawal-associated environmental cues. The average percentage of c-Fos positive neurons labeled with FG in the chronic morphine + naloxone group was 16.3 ± 1.0%, which was significantly higher than that in the saline + saline (11.3 ± 0.7%), saline + naloxone (11.8 ± 1.0%), or chronic morphine + saline (11.1 ± 1.6%) groups (n = 6, one-way ANOVA, Bonferroni post hoc analysis, F
(3, 20) = 4.482, P = 0.03, Fig. 9c). This result suggests that withdrawal-associated environmental cues can activate mPFC-to-BLA projection neurons in morphine-withdrawal rats.
Overexpression of miR-105 in the mPFC leads to suppression of D1 receptor expression in glutamatergic terminals of the projection neurons from the mPFC to the BLA, and a reduction in CPA in morphine-withdrawal rats
To test the functional relevance of the morphine-induced suppression of miR-105 in the mPFC we opposed this by intra-mPFC injection of a LV containing a miR-105 precursor, injecting a control group of rats with the eGFP-labeled LV alone. This intervention was performed 3–4 weeks before morphine administration and we found that over-expressing miR-105 in the mPFC by these means significantly decreased the expression of D1 receptors in glutamatergic terminals of projection neurons from the mPFC to the BLA (relative to the control group), as assessed after morphine treatment using the triple immunofluorescence staining method. The top and bottom panels of Fig. 10a show D1R staining (column 1, Fig. 10a, red color), eGFP labeling (column 2, Fig. 10a, green color), glutamatergic terminal marker VGLUT2 immunoreactivity (column 3, Fig. 10a, blue color), and coexpression of D1R, eGFP, and VGLUT2 (column 4, Fig. 10a, white color) in slices of the BLA in the control group of rats injected with LV alone (miR-105 NC-LV) and the group injected with the miR-105 precursor (miR-105-LV). The average percentage of the normalized D1 receptor intensity in glutamatergic terminals of projection neurons from the mPFC to the BLA was 100.0 ± 10.8% in the miR-105 NC-LV group (n = 5) and 59.5 ± 8.3% in the miR-105-LV group (n = 5, independent t test, P = 0.018, compared to miR-105 NC-LV group, Fig. 10b). We then examined the influence of over-expressing miR-105 on environmental cue-induced place aversion in morphine-withdrawal rats. The results showed that, in the group of rats that had previously received the intra-mPFC injection of miR-105 overexpression lentivirus, the post-conditioning test CPA score was significantly lower (miR-105-LV group, −18.7 ± 23.9 s, n = 6) than that in the control group injected with the LV alone (miR-105 NC-LV group, −193.8 ± 35.1 s, n = 6) (two-way ANOVA, Bonferroni post hoc analysis, F
(1, 21) = 8.67, P = 0.007, compared to post-conditioning test CPA score in miR-105 NC-LV group, Fig. 10c). This result suggests that a chronic morphine-induced increase in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA contributes to CPA in morphine-withdrawal rats. Nevertheless, miR-105 also has other targets that may affect these results. However, the present known targets of miR-105 are mainly those involved in tumor behavior and malignant progression [27,28,29] such as mRNAs of SOX9, SUZ, and NCOA1. Moreover, the results presented in Fig. 6 herein showed that a chronic morphine-induced increase in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA resulted in a sensitization of the effect of D1 receptor agonist on glutamate release. Further, our pervious study showed that the intra-BLA injection of D1 receptor antagonist significantly inhibited CPA in morphine-withdrawal rats [8]. This evidence further supports the notion that D1 receptors in glutamatergic terminals of projection neurons from the mPFC to the BLA contribute to CPA in morphine-withdrawal rats, although we cannot completely eliminate the contributions of other targets of miR-105 in this behavior.