Article Figures & Data
Figures
- Figure 1.
Knock-down of mGluR5 in striatal dopamine receptor D1-MSNs. A, Design of the transgene expressing GFP as a marker and two interfering RNAs (iRNAs). This construct was inserted after the translational start of the gene encoding the dopamine D1 receptor in a bacterial artificial chromosome. B, Sequences of iRNAs. Interfering sequence is depicted in bold. Red arrows indicate targeted regions of mGluR5 mRNA. C, Expression of the transgene in mGluR5KD-D1 mice (KD) as detected by immunohistochemistry for GFP in a sagittal brain section. Higher magnification showing difference between staining of cell bodies in the caudate–putamen (CPu) and its projections to ventral midbrain nuclei (VMN). D, The transgene (GFP; green) is expressed in ∼53% of the striatal neurons (NeuN; red; → indicates examples of GFP-positive neurons and ▶ indicates examples of GFP-negative neurons). E, The expression of the construct is selective for D1-MSNs. Thus, expression is limited to MSNs (DARPP-32; blue) and absent from D2-MSNs (labeled by red immunofluorescent labeling of prepro enkephalin; ppEnk). Examples of GFP-expressing (→) and non-GFP-expressing (▶) MSNs. F, Expression of mGluR5 in the striatum as shown with in situ hybridization. G, Knock-down assessment by quantitative PCR (n = 4–5, p < 0.001) and H, Western-blotting with representative blot example shown (n = 4, p = 0.0112). Data are presented as mean + SEM, p-value of t test (*p < 0.05, **p < 0.001). Scale bars 20 μm. Cx, Cortex; Acb, nucleus accumbens.
- Figure 2.
Knock-down of mGluR5 does not interfere with other similar transcripts or with the production of endogenous microRNAs. A, Microarray analysis showing that the transgene did not alter the expression levels of other metabotropic glutamate receptor family members nor the levels of the related GABAB1 receptor in mGluR5KD-D1 mice (KD). B, The yield of small RNAs (<200 nt) isolated from the striatum of transgenic mice was normal. C, The abundance of the mature form of eight randomly selected endogenous miRNAs was not altered, indicating an intact microRNA processing machinery. *p < 0.005.
- Figure 3.
Cocaine self-administration and cue-induced reinstatement in mGluR5KD-D1 (KD) and control (WT) mice. Lever-press responses during cocaine self-administration (A, B), extinction (C), and cue-induced reinstatement test phases (D). A, Self-administration: cocaine-reinforced (•, active) and nonreinforced (▴, inactive) responses across five different doses of cocaine did not differ between genotypes (lever × genotype × dose interaction, F(4,102) = 0.125; p > 0.05). B, Similarly, lever responses across 10 consecutive sessions with a 0.75 mg/kg per infusion training dose did not significantly differ between WT and KD mice (session × lever × genotype, F(9,198) = 1.56; p > 0.05). During the training phase, the presentation of a CS was associated with each cocaine infusion. C, Extinction: responses on the active lever during the last 3 sessions of cocaine self-administration (C3-1) and 14 subsequent extinction sessions did not differ between the two genotypes (session × genotype, F(16,288) = 1.27; p > 0.05). D, Reinstatement: lever responses during the last extinction session (Ext) and the cue-induced reinstatement test (Reinst). Contingent presentation of the CS increased the number of responses on the active lever over extinction performance, in mice from both genotypes. However, reinstatement of the cocaine-seeking response was significantly lower in KD mice (genotype × lever × condition, F(1,36) = 5.12; p < 0.05). *Post hoc significant difference (p < 0.05) from WT. #Significant difference (p < 0.01) from the active lever responses during extinction. §Significant difference (p < 0.01) from active lever responses during reinstatement. Responses are plotted as mean (±SEM).
- Figure 4.
Associative learning in mGluR5KD-D1 (KD) and control (WT) mice. A, Pavlovian conditioning: entries into a food magazine increased during presentations of a stimulus associated with food delivery (CS+), but decreased during presentations of a stimulus associated with no outcome (CS−). B, Goal-tracking: magazine entries that occurred following CS+ onset, but before food delivery (that is, goal-tracking responses), significantly increased across conditioning sessions (main effect of session, F(10,190) = 7.6, p < 0.01), but did not differ between genotypes (session × genotype interaction, F(10,190) = 0.171, p > 0.05). C, Food self-administration for PIT cohort: both genotypes responded more on a lever that resulted in food delivery (Act), than an alternate lever on which responding had no consequence (Ina), when food delivery occurred under an FR1 (main effect of lever, F(1,14) = 54.84, p < 0.001; lever × genotype interaction F(1,14) = 1.18, p > 0.05) or a variable-interval 60 s schedule (VI60) (main effect of lever F(1,14) = 37.61, p < 0.001; lever × genotype F(1,14) = 0.10, p > 0.05). D, PIT test: responses on a lever that previously led to the delivery of food significantly increased during CS+ presentations, compared with a decrease in responding during CS− presentations (main effect of stimulus, F(1,14) = 20.93, p < 0.001). There was no difference in PIT between genotypes (stimulus × genotype, F(1,14) = 0.125, p > 0.05). Elevation score = lever responses during CS minus responses pre CS. E, Conditioned reinforcement: both genotypes preferentially responded on a lever that led to CS+ presentations, compared with a CS− paired lever (main effect of lever, F(1,19) = 24.38, p < 0.001). However, KD mice made significantly fewer CS+ paired lever responses than WT mice (genotype × lever, F(1,19) = 5.57, p < 0.05). *p < 0.05, post hoc comparison between genotypes by t test. F, Sign-tracking: both genotypes preferentially approached the location of the CS+ during its presentations. However, KD mice made significantly fewer CS+ approaches than WT mice. #p < 0.05, comparison between genotypes by Wilcoxon matched pairs test.
