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Cocaine exposure reorganizes cell type– and input-specific connectivity in the nucleus accumbens

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Abstract

Repeated exposure to cocaine alters the structural and functional properties of medium spiny neurons (MSNs) in the nucleus accumbens (NAc). These changes suggest a rewiring of the NAc circuit, with an enhancement of excitatory synaptic connections onto MSNs. However, it is unknown how drug exposure alters the balance of long-range afferents onto different cell types in the NAc. Here we used whole-cell recordings, two-photon microscopy, optogenetics and pharmacogenetics to show how repeated cocaine exposure alters connectivity in the mouse NAc medial shell. Cocaine selectively enhanced amygdala innervation of MSNs expressing D1 dopamine receptors (D1-MSNs) relative to D2-MSNs. We also found that amygdala activity was required for cocaine-induced changes to behavior and connectivity. Finally, we established how heightened amygdala innervation can explain the structural and functional changes evoked by cocaine. Our findings reveal how exposure to drugs of abuse fundamentally reorganizes cell type– and input-specific connectivity in the NAc.

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Figure 1: Repeated cocaine exposure enhances excitatory connectivity.
Figure 2: Synaptic plasticity is both cell type– and input-specific.
Figure 3: Changes to the number and strength of connections.
Figure 4: Alterations to structural connections at dendritic spines.
Figure 5: Synaptic reorganization depends on NMDARs.
Figure 6: Enhancement of BLA inputs depends on BLA activity.
Figure 7: Enhanced connectivity reflects BLA innervation.

Change history

  • 19 August 2014

    In the version of this article initially published online, the top of the y axis in the right subpanel of Figure 1e was labeled 100. The correct value is 10. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Carter laboratory, M. Farrant and R. Froemke for helpful discussions and comments on the manuscript, P. Kennedy for advice on cocaine administration, and C. Farb, R. Sears and H. Seong for help with anatomy. This work was supported by the Wellcome Trust (A.F.M.), and the Dana Foundation, McKnight Foundation and US National Institutes of Health R01DA038138 (A.G.C.).

Author information

Authors and Affiliations

Authors

Contributions

A.F.M. and A.G.C. designed the experiments. A.F.M. performed the experiments and analyzed the data. J.M.C. performed the stereotaxic injections. A.F.M. and A.G.C. wrote the paper.

Corresponding author

Correspondence to Adam G Carter.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Development of behavioral sensitization and viral injection sites.

a. Locomotion of saline (grey, top) or cocaine (black, bottom) mice, measured for 10 min immediately after injection on day 1 (left) and day 5 (right). Scale bar = 5 cm.

b. Summary of time course of behavioral sensitization. On day 0, all mice received a saline injection; on days 1 – 5, mice received either an injection of saline (grey) or 15 mg / kg cocaine (black); on day 8, all mice received a challenge injection of 15 mg / kg cocaine. Note that the effect of cocaine increases after each injection (cocaine day 1 vs. day 5: t(10) = 5.0, p = 0.001, paired t-test; saline vs. cocaine day 8: U = 14, p = 0.006, Mann-Whitney; n = 9 (s), 11 (c) mice). Summary shows mean ± SEM. * denotes p < 0.05.

c. Confocal images showing ChR2 expression at viral injection site in BLA (left), VH (center) and PFC (right). Scale bar = 100 μm.

Supplementary Figure 2 BLA and VH EPSCs at multiple stimulus intensities.

a. Average light-evoked BLA EPSCs recorded at neighboring D1‑MSNs (black) and D2‑MSNs (red) on day 8 from animals injected with saline on days 1 to 5. The duration of light pulse was varied between 0.5 ms and 2 ms. Darker colors show longer stimulus durations. Scale bar = 50 pA, 20 ms.

b. Summary of BLA EPCSs at D1-MSNs (black) and D2-MSNs (red). In saline conditions, there is no difference between the two cell types at any light intensity.

c, d. Similar to (a, b) but for mice injected with cocaine on days 1 to 5. After repeated cocaine exposure, D1‑MSNs have larger BLA EPSCs than neighboring D2-MSNs.

e. Summary of D1/D2 ratio for BLA EPSCs recorded in response to 1 ms light pulse in animals treated with either saline (s) or cocaine (c) (D1/D2 ratio: U = 5, p = 0.01, Mann-Whitney; n = 8 (s), 7 (c) pairs).

f - j. Similar to (a - e), but for VH EPSCs. Note that VH input is larger onto D1-MSNs in saline conditions, but this difference is no longer apparent after cocaine treatment (D1/D2 ratio: U = 11, p = 0.01, Mann-Whitney; n = 8 (s), 10 (c) pairs).

Summary shows mean ± SEM. * denotes p < 0.05.

Supplementary Figure 3 Repeated exposure to cocaine does not change PFC D1/D2 ratio.

a. (Top) Schematic of AAV9-CAG-hChR2-mCherry injection in medial prefrontal cortex (PFC, grey) and projections to the NAc. (Bottom) Coronal section of the NAc medial shell (dotted outline), showing distribution of PFC afferents. Scale bar = 100 μm.

b. Light-evoked PFC EPSCs at D1‑MSNs (mean in black, error in grey) and D2‑MSNs (mean in red, error in pink) on day 8 from animals injected with saline (left) or cocaine (right) on days 1 to 5. EPSCs at D1‑MSNs are normalized to those at neighboring D2‑MSNs. Scale bar = unity, 20 ms.

c. (Left) Summary of PFC EPSCs at D1-MSNs and D2-MSNs in saline (top) and cocaine (bottom) mice. (Right) Summary of D1/D2 ratio of PFC EPSCs. Note that cocaine does not alter the D1/D2 ratio of PFC EPSCs (D1/D2 ratio: U = 15, p = 0.7, Mann-Whitney; n = 6 pairs).

Summary shows mean ± SEM. * denotes p < 0.05.

Supplementary Figure 4 Repeated exposure to cocaine does not alter BLA or VH PPR.

a. Schematic of recording configuration showing neighboring D1‑MSNs (black) and D2‑MSNs (red) receiving BLA input. Afferent fibers were stimulated at distal locations in order to avoid confounds due to direct excitation of ChR2 in the presynaptic terminal.

b. Light-evoked BLA EPSCs at D1‑MSNs (black) and D2‑MSNs (red) on day 8 from animals injected with saline (left) or cocaine (right) on days 1 to 5. Inter-stimulus interval = 50 ms.

c. Summary of paired-pulse ratio (PPR) for BLA EPSCs at D1-MSNs and D2-MSNs in saline and cocaine mice (no significant effects, two-way ANOVA).

d. Summary of D1/D2 ratio of BLA PPR. Note that cocaine has no effect on BLA PPR (D1/D2 ratio: U = 27, p = 0.96, Mann-Whitney; n = 8 (s), 7 (c) pairs).

e - h. Similar to (a - d) but for VH input. Note that cocaine has no effect on VH PPR (no significant effects, two-way ANOVA; D1/D2 ratio: U = 21, p = 1, Mann-Whitney; n = 6 (s), 7 (c) pairs).

Summary shows mean ± SEM. * denotes p < 0.05.

Supplementary Figure 5 Synaptic calcium signals at dendritic spines.

a. Two-photon image of dendrites and spines of MSN filled with Alexa Fluor 594 and Fluo-4FF via the patch pipette, before (left) and after (right) two-photon glutamate uncaging. Orange circles show uncaging location. Note easily detectable calcium signal. Scale bar = 2 μm.

b. Summary of amplitudes of uncaging-evoked calcium signals at D1-MSNs (black) and D2-MSNs (red) with increasing distance from the soma (left) or with increasing spine volume (right). Note that all spines have detectable calcium signals, showing no change in amplitude with either distance along the dendrite or spine volume.

c. Summary of sampled dendrite locations for BLA (purple) and VH (blue) two-photon mapping experiments in saline (s) and cocaine (c) mice (D1/D2 ratio: BLA: U = 11, p = 0.54, n = 6 (s), 5 (c) pairs; VH: U = 11, p = 0.84, n = 5 (s), 5 (c) pairs; Mann-Whitney). Original data is shown in Fig. 4.

Summary shows mean ± SEM. * denotes p < 0.05.

Supplementary Figure 6 Additional MK-801 behavior experiments.

a. (Left) Schematic of experimental protocol. Mice were pretreated with MK-801 or saline 30 min before each saline injection on days 1 to 5, followed by a challenge injection of cocaine on day 8 to monitor behavior. (Right) Summary of distance traveled by saline- (s) and MK-801- (m) pretreated mice after the challenge injection of cocaine on day 8 (U = 14, p = 0.93, Mann-Whitney; n = 5 (s), 6 (m) mice).

b. (Left) Schematic of experimental protocol. Mice were pretreated with MK-801 or saline 30 min before a cocaine injection on days 1 to 5, followed by a baseline locomotion measurement on day 8. (Right) Summary of distance traveled by saline- (s) and MK‑801- (m) pretreated mice before any cocaine challenge on day 8 (U = 29, p = 0.54, Mann-Whitney; n = 8 (s), 9 (m) mice).

c. (Left) Schematic of experimental protocol. Mice were pretreated with MK-801 or saline 30 min before a challenge injection of cocaine on day 8. (Right) Summary of distance traveled by saline- (s) and MK-801- (m) pretreated mice after the challenge injection of cocaine on day 8 (U = 5, p = 0.1, Mann-Whitney; n = 5 (s), 6 (m) mice).

Summary shows mean ± SEM. * denotes p < 0.05.

Supplementary Figure 7 Characterization of DREADDs in the BLA.

a. Wide-field fluorescence image showing expression of mCitrine after viral injection of AAV8-CaMKIIa-HA-hM4D-IRES-mCitrine into the BLA. Scale bar = 100 μm.

b. Wide-field fluorescence image showing lack of expression of mCitrine in the VH after viral injection into the BLA. Scale bar = 100 μm.

c. Confocal image showing expression of hM4D-positive axons in the NAc after viral injection into the BLA. Scale bar = 10 μm.

d. Current-clamp recording from an hM4D-expressing BLA neuron, with current injection eliciting action potential firing before but not after bath application of 10 μM CNO. Scale bar = 20 mV, 400 ms.

e. Summary of effect of CNO on action potential firing at hM4D-expressing BLA neurons (t(4) = 4.5, p = 0.01, paired t-test; n = 5 cells).

f. Wide-field fluorescence image showing expression of mCitrine (green) and ChR2-mCherry (red) after viral injections into the BLA. Scale bar = 300 μm.

g. Enlargement of boxed region in (f), showing overlapping expression of mCitrine and ChR2-mCherry. Scale bar = 100 μm.

h. Confocal images showing co-expression of hM4D (green) and ChR2-mCherry (red) in BLA neurons. Scale bar = 20 μm.

I, j. Quantification of neurons in the BLA infected with ChR2 alone (red), hM4D alone (green), and both ChR2 and hM4D (orange).

Supplementary Figure 8 Additional BLA DREADD behavior experiments.

a. (Left) Schematic of experimental protocol. Mice were injected with AAVs to express hM4D bilaterally in the basolateral amygdala (BLA). Two weeks later, mice were pretreated with saline 30 min before each cocaine injection on days 1 to 5, followed by a challenge injection of cocaine on day 8 to monitor behavior. (Right) Summary of distance traveled by control or hM4D-expressing mice after cocaine challenge injection on day 8 (U = 20, p = 0.62, Mann-Whitney; n = 7 (Ctrl), 7 (hM4D) mice).

b. (Left) Schematic of experimental protocol. Mice were injected with AAVs to express hM4D bilaterally in the basolateral amygdala (BLA). Two weeks later, mice were pretreated with CNO 30 min before each saline injection on days 1 to 5, followed by a challenge injection of cocaine on day 8 to monitor behavior. (Right) Summary of distance traveled by control or hM4D-expressing mice after cocaine challenge injection on day 8 (U = 10, p = 0.69, Mann-Whitney; n = 5 (Ctrl), 5 (hM4D) mice).

c. (Left) Schematic of experimental protocol. Mice were injected with AAVs to express hM4D bilaterally in the basolateral amygdala (BLA). Two weeks later, mice were pretreated with CNO 30 min before each cocaine injection on days 1 to 5, followed by a baseline locomotion measurement on day 8. (Right) Summary of distance traveled by control or hM4D-expressing mice before any cocaine challenge on day 8 (U = 15, p = 0.28, Mann-Whitney; n = 6 (Ctrl), 8 (hM4D) mice).

Summary shows mean ± SEM. * denotes p < 0.05.

Supplementary Figure 9 Characterization of DREADDs in the VH.

a. Wide-field fluorescence image showing expression of mCitrine after viral injection of AAV8-CaMKIIa-HA-hM4D-IRES-mCitrine into the VH. Scale bar = 100 μm.

b. Wide-field fluorescence image showing lack of expression of mCitrine in the BLA after viral injection into the VH. Scale bar = 100 μm.

c. Confocal image showing expression of hM4D-positive axons in the NAc after viral injection into the VH. Scale bar = 10 μm.

d. Current-clamp recording from an hM4D-expressing VH neuron, with current injection eliciting action potential firing before but not after bath application of 10 μM CNO. Scale bar = 20 mV, 200 ms.

e. Summary of effect of CNO on action potential firing at hM4D-expressing VH neurons (t(4) = 3.8, p = 0.02, paired t-test; n = 5 cells).

f. Wide-field fluorescence image showing expression of mCitrine (green) and ChR2-mCherry (red) after viral injections into the VH. Scale bar = 300 μm.

g. Enlargement of boxed region in (f), showing overlapping expression of mCitrine and ChR2-mCherry. Scale bar = 100 μm.

h. Confocal images showing co-expression of hM4D (green) and ChR2-mCherry (red) in VH neurons. Scale bar = 20 μm.

i, j. Quantification of neurons in the VH infected with ChR2 alone (red), hM4D alone (green), and both ChR2 and hM4D (orange).

Supplementary Figure 10 Enhancement of VH inputs depends on VH activity.

a. Schematic of experimental protocol. Mice were injected with AAVs to express hM4D bilaterally in the ventral hippocampus (VH). Two weeks later, mice were pretreated with CNO 30 min before each cocaine injection on days 1 to 5, followed by a cocaine challenge on day 8 to monitor behavioral sensitization (b), or electrophysiology on day 8 without a cocaine challenge to assess synaptic connectivity (c - f).

b. (Left) Locomotion of control (top) or hM4D-expressing (bottom) mice, measured for 10 min immediately after a challenge injection of cocaine on day 8. Scale bar = 5 cm. (Right) Summary of distance traveled by control or hM4D-expressing mice after the cocaine challenge. Dashed line indicates saline control. Note that inhibiting VH activity does not prevent behavioral sensitization (U = 17, p = 0.23, Mann-Whitney; n = 8 (Ctrl), 7 (hM4D) mice).

c. (Top) Schematic of AAV injection to express hM4D or control GFP bilaterally in the VH, and ChR2 unilaterally in the BLA. (Bottom) Light-evoked BLA EPSCs at D1‑MSNs (black) and D2‑MSNs (red) from control (left) and hM4D-expressing (right) mice. EPSCs at D1‑MSNs are normalized to those at neighboring D2‑MSNs (see Figure 2). Scale bar = unity, 20 ms.

d. (Left) Summary of BLA EPSCs at D1-MSNs (top) and D2-MSNs (bottom) in control (top) and hM4D-expressing (bottom) mice. (Right) Summary of D1/D2 ratio of BLA EPSCs. Note that inhibiting VH activity does not prevent the biasing of BLA EPSCs onto D1-MSNs (D1/D2 ratio: U = 23, p = 0.38, Mann-Whitney; n = 8 (Ctrl), 8 (hM4D) pairs).

e-f. Similar to (c - d), with hM4D or control GFP expressed bilaterally in the VH, and ChR2 expressed unilaterally in the VH. Note that inhibiting VH activity reverses the normalization of VH ESPCs at D1- and D2-MSNs (D1/D2 ratio: U = 23, p = 0.001, Mann-Whitney; n = 11 (Ctrl), 15 (hM4D) pairs).

Bar graphs show mean ± SEM. * denotes p < 0.05.

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MacAskill, A., Cassel, J. & Carter, A. Cocaine exposure reorganizes cell type– and input-specific connectivity in the nucleus accumbens. Nat Neurosci 17, 1198–1207 (2014). https://doi.org/10.1038/nn.3783

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