Cue- versus reward-encoding basolateral amygdala projections to nucleus accumbens
- Departments of Neuroscience, University of Pittsburgh, United States
- Departments of Psychiatry, University of Pittsburgh, United States
Figures
Figure 1
R-spondin2-positive (Rspo2)- and protein phosphatase 1 regulatory subunit 1B-positive (Ppp1r1b)-to-nucleus accumbens (NAc) projections.
(A) Schematic for breeding Rspo2-Cre × D1-tdTomato mice using heterozygous Rspo2-Cre and D1-tdTomato mice. (B) Example agarose gel image showing four genotypes of bred mice. (C) Diagram showing injection of AAV-DIO-channelrhodopsin-2 (ChR2) into the basolateral amygdala (BLA) of Rspo2-Cre mice. (D, E) Example bright-field (D) and fluorescence (E) images showing ChR2-eYFP expression in the BLA. (F, G) Example bright-field (F) and fluorescence (G) images showing ChR2-eYFP expression in Rspo2 presynaptic terminals within the NAc. (H, I) Enlarged portions of (G) showing Ch2R-expressing fibers in the NAc core (H) and shell (I). (J) Diagram showing patch-clamp recordings of NAc D1 and D2 medium spiny neurons (MSNs) in response to optogenetic stimulation of Rspo2-to-NAc projection in brain slice. (K, L) Examples (K) and summary (L) showing that excitatory postsynaptic currents (EPSCs) evoked in D1 and D2 MSNs by optogenetic stimulation of ChR2-expressing Rspo2 presynaptic fibers were inhibited by NBQX (10 µM) and D-AP5 (50 µM) (D1, p<0.01 basal vs. NBQX+AP5; D2, p<0.01 basal vs. NBQX+AP5, two-tailed t-test). (M) Schematic for breeding Cartpr-Cre × D1-tdTomato mice. (N) Example gel image showing the genotyping of bred mice. (O) Diagram showing injection of AAV-DIO-ChR2 into the BLA of cocaine- and amphetamine-regulated transcript protein (Cartpt)-Cre mice. (P, Q) Example bright-field (P) and fluorescence (Q) images showing expression of ChR2-eYFP in the BLA. (R, S) Example bright-field (R) and fluorescence (S) images showing the ChR2-eYFP-expressing axon terminals in the NAc. (T, U) Enlarged portions of (S) showing Ch2R-expressing fibers in the NAc core (T) and shell (U). (V) Diagram showing recordings of NAc D1 and D2 MSNs in response to optogenetic stimulation of Ppp1r1b-to-NAc projection. (W, X) Examples (W) and summary (X) showing that EPSCs elicited in D1 and D2 MSNs by optogenetic stimulation of ChR2-expressing Ppp1r1b presynaptic fibers were inhibited by NBQX (10 µM) and D-AP5 (50 µM) (D1, p=0.01 basal vs. NBQX+AP5; D2, p<0.01 basal vs. NBQX+AP5, two-tailed t-test). (Y) Example EPSCs from Rspo2- or Ppp1r1b-to-NAc synapses in D1 and D2 MSNs evoked by paired-pulse stimulations with different interpulse intervals (25, 50, 100, and 200 ms). (Z) Summary showing lack of difference in the paired-pulse ratio between D1- and D2-MSNs within projections (Rspo2 D1 vs. Rspo2 D2, F1, 10 = 0.3, p=0.61; Ppp1r1b D1 vs. Ppp1r1b D2, F1, 10 = 0.0, p=0.83; two-way mixed ANOVA). n/m, number of recorded cells/number of mice.
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Figure 1—source data 1
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- https://cdn.elifesciences.org/articles/89766/elife-89766-fig1-data1-v1.xlsx
Figure 2
Optogenetic intracranial self-stimulation (ICSS) of R-spondin2-positive (Rspo2)- and protein phosphatase 1 regulatory subunit 1B-positive (Ppp1r1b)-to-nucleus accumbens (NAc) presynaptic terminals.
(A) Diagrams showing intra-NAc optogenetic stimulation of Rspo2- or Ppp1rb-to-NAc presynaptic terminals during ICSS (upper) and the optogenetic ICSS setup (lower). (B, C) Summaries showing that intra-NAc stimulation at 20 or 40 Hz did not establish ICSS in control mice, in which basolateral amygdala (BLA) Rspo2-to-NAc (active vs inactive, F1,6 = 0.1, p=0.76, two-way mixed ANOVA, B) or Ppp1r1b-to-NAc (active vs inactive, F1, 6 = 0.0, p=0.88, two-way mixed ANOVA, C) presynaptic terminals expressed YFP. (D, E) Summaries showing that intra-NAc optogenetic stimulation of channelrhodopsin-2 (ChR2)-expressing Rspo2- (20 Hz, active vs inactive, F1,6 = 2.8, p=0.14, two-way mixed ANOVA; 40 Hz, active vs inactive, F1,6 = 6.3, p=0.04, two-way mixed ANOVA, D) or Ppp1r1b-to-NAc (20 Hz, active vs inactive, F1,6 = 0.9, p=0.37, two-way mixed ANOVA; 40 Hz, active vs inactive, F1,6 = 2.6, p=0.16, two-way mixed ANOVA, E) presynaptic terminals at 40 Hz, but not 20 Hz, established ICSS. (F, G) Summaries showing that, compared with Rspo2-ChR2 mice (YFP vs ChR2 main effect, F1, 12 = 2.1, p=0.17, three-way ANOVA, F) Ppp1r1b-ChR2 mice (YFP vs ChR2 main effect, F1,12 = 8.9, p=0.01, three-way ANOVA, G) exhibited persistent cue-induced operant ,responses during the extinction test (lever press resulting in cue presentation without optogenetic stimulation) after cue-conditioned ICSS was established. (H, I) Summaries showing that after 21-day abstinence following the ICSS establishment, cue-induced responding to the active lever was minimal in Rspo2-ChR2 mice (ChR2-active vs ChR2-inactive, t=0.8, p=0.48, n=4, two-tailed t-test, H) but robust in Ppp1r1b-ChR2 mice (ChR2-active vs ChR2-inactive, t=2.9, p=0.03, n=4, two-tailed t-test, I).
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Figure 2—source data 1
Source data containing numerical raw data of all panels.
- https://cdn.elifesciences.org/articles/89766/elife-89766-fig2-data1-v1.xlsx
Figure 3
Roles of R-spondin2-positive (Rspo2)- and protein phosphatase 1 regulatory subunit 1B-positive (Ppp1r1b)-to-nucleus accumbens (NAc) transmissions in acquisition of cocaine self-administration.
(A, B) Diagrams showing viral expression of stabilized step function opsin (SSFO) in basolateral amygdala (BLA) Rspo2 or Ppp1r1b neurons (A) and intra-NAc optical stimulation (B). (C) Example spontaneous excitatory postsynaptic currents (sEPSCs) in NAc medium spiny neurons (MSNs) during baseline recording, after 473 nm laser stimulation, and after 590 nm laser stimulation of SSFO-expressing presynaptic terminals. (D) Summaries showing that stimulation of SSFO-expressing presynaptic terminals by 473 nm laser induced an increase in sEPSC frequency, and this increase was reversed by 590 nm laser (F2,10 = 6.0, p=0.02, one-way ANOVA repeated measure). (E) Timeline for SSFO-mediated manipulation of Rspo2- or Ppp1r1b-to-NAc transmission during cocaine self-administration. (F, G) Summaries showing that numbers of active lever presses and optical stimulations during cocaine self-administration were not altered upon SSFO-mediated upregulation of Rspo2-to-NAc (active lever press, F4,36 = 1.2, p=0.32; inactive lever press, F4,36 = 1.5, p=0.22; infusion, F4,36 = 1.5, p=0.21; one-way ANOVA repeated measure, F) or Ppp1r1b-to-NAc (active lever press, F4,20 = 1.5, p=0.25; inactive lever press, F4,20 = 0.4, p=0.87; infusion: F4,20 = 1.8, p=0.16, Tukey posttest, G), compared to data 1 day before and 1 day after the day with SSFO stimulation. (H, I) Diagrams showing viral expression of hM4D DREADDs in BLA Rspo2 or Ppp1r1b neurons (H) and intra-NAc infusion of clozapine N-oxide (CNO) (I). (J) Experimental schematic of intra-NAc infusion of CNO or saline control during daily cocaine self-administration. Inset: Symbols of mice with different manipulations in K–N. (K, L) Summaries showing that chemogenetic inhibition of Rspo2-to-NAc transmission did not alter numbers of active lever presses (saline vs CNO, F1, 24 = 0.4, p=0.53, two-way mixed ANOVA, K) or numbers of cocaine infusions (saline vs CNO, F1, 24 = 0.4, p=0.52, two-way mixed ANOVA, L). (M, N) Summaries showing that chemogenetic inhibition of Ppp1r1b-to-NAc transmission did not alter numbers of active lever presses (saline vs CNO, F1, 24 = 0.3, p=0.61, two-way mixed ANOVA, M) or numbers of cocaine infusions (saline vs CNO, F1, 24 = 0.1, p=0.71, two-way mixed ANOVA, N).
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Figure 3—source data 1
Source data containing numerical raw data of all panels.
- https://cdn.elifesciences.org/articles/89766/elife-89766-fig3-data1-v1.xlsx
Figure 4
Roles of protein phosphatase 1 regulatory subunit 1B-positive (Ppp1r1b)- and R-spondin2-positive (Rspo2)-to-nucleus accumbens (NAc) transmissions in cue-induced cocaine seeking.
(A, B) Diagram showing viral expression of DREADDs in basolateral amygdala (BLA) Rspo2 or Ppp1r1b neurons (A) and intra-NAc application of clozapine N-oxide (CNO) (B). ( C–J) Example bright-field and fluorescence images of the BLA and NAc in mice with Cre-dependent intra-BLA expression of DREADDs in Rspo2-Cre (C–F) and cocaine- and amphetamine-regulated transcript protein (Cartpt)-Cre (G–J) mice. (K) Schematic of cocaine self-administration and DREADDs-mediated manipulation of Rspo2- or Ppp1r1b-to-NAc subprojection during cue-induced cocaine seeking after withdrawal from cocaine self-administration. (L) Collection of symbols indicating mice with different manipulations in M–X. (M–R) Summaries showing that, after 10 days of cocaine self-administration, cue-induced cocaine seeking (assessed by numbers of active lever presses) was not altered in Rspo2-hM4D mice (M–O) but decreased in Ppp1r1b-hM4D (P–R) mice upon intra-NAc application of CNO (Rspo2: saline vs CNO, F1,15 = 0.6, p=0.45; Ppp1r1b: saline vs CNO, F1,10 = 7.1, p=0.02; two-way mixed ANOVA main effect). (S–X) Summaries showing that, after 10 days of cocaine self-administration, cue-induced cocaine seeking was not altered in Rspo2-hM3D mice (M–O) but increased in Ppp1r1b-hM3D (P–R) mice upon intra-NAc application of CNO (Rspo2: saline vs CNO, F1,12 = 1.6, p=0.23; Ppp1r1b: saline vs CNO, F1,10 = 7.4, p=0.02; two-way mixed ANOVA main effect).
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Figure 4—source data 1
Source data containing numerical raw data of all panels.
- https://cdn.elifesciences.org/articles/89766/elife-89766-fig4-data1-v1.xlsx
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Cue- versus reward-encoding basolateral amygdala projections to nucleus accumbens
eLife 12:e89766.
https://doi.org/10.7554/eLife.89766
