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Spine pruning drives antipsychotic-sensitive locomotion via circuit control of striatal dopamine

Nature Neuroscience volume 18, pages 883–891 (2015)Cite this article

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Abstract

Psychiatric and neurodevelopmental disorders may arise from anomalies in long-range neuronal connectivity downstream of pathologies in dendritic spines. However, the mechanisms that may link spine pathology to circuit abnormalities relevant to atypical behavior remain unknown. Using a mouse model to conditionally disrupt a critical regulator of the dendritic spine cytoskeleton, the actin-related protein 2/3 complex (Arp2/3), we report here a molecular mechanism that unexpectedly reveals the inter-relationship of progressive spine pruning, elevated frontal cortical excitation of pyramidal neurons and striatal hyperdopaminergia in a cortical-to-midbrain circuit abnormality. The main symptomatic manifestations of this circuit abnormality are psychomotor agitation and stereotypical behaviors, which are relieved by antipsychotics. Moreover, this antipsychotic-responsive locomotion can be mimicked in wild-type mice by optogenetic activation of this circuit. Collectively these results reveal molecular and neural-circuit mechanisms, illustrating how diverse pathologies may converge to drive behaviors relevant to psychiatric disorders.

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Figure 1: Arp2/3 mutant mice respond to antipsychotics and exhibit elevated stDA.
Figure 2: Regional rescue implicates the frontal cortex in mediating the elevated motor activity and striatal dopaminergic tone of the Arp2/3 mutant mice.
Figure 3: Arp2/3-rescued excitatory neurons of the frontal cortex project to and make synaptic contacts in the VTA/SNc.
Figure 4: Loss of Arp2/3 function leads to the formation of abnormal synaptic contacts.
Figure 5: Spine loss leads to excitation of the cortico-VTA/SNc circuit in Arp2/3 mutant mice.
Figure 6: Selective activation of the cortico-VTA/SNc circuit in WT mice stimulates haloperidol-sensitive locomotion and elevates stDA.

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  • 07 May 2015

    In the version of this article initially published online, "USA" was included at the end of the two affiliations in Hungary. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank M. Caron, B. Hogan and C. Eroglu for critical reading and comments. We also thank K. Kobayashi (Fukushima Medical University, Japan) for providing the FuGB2 viral vector, K. Sakurai (Duke University) for providing the hSyn-Cre lentiviral vector, and R. Rodriguiz and E. Spence for behavioral technical support. This work was supported by the following grants: US National Institutes of Health (NIH) MH103374 and NS059957 (S.H.S.), NIH NS077986 (F.W.), AA021074 (H.Y.), NS039444 (R.J.W.) and MH082441 (W.C.W.). M.R. is supported by a US National Research Foundation fellowship and B.R. is supported by the János Bólyai Research Fellowship from the Hungarian Academy of Sciences, by the Hungarian Scientific Research Fund (OTKA, grant K83830) and by the Szent István University, Faculty of Veterinary Science (Research Faculty Grant 2014). Some of the experiments were conducted with equipment/software purchased with a North Carolina Biotechnology Center grant (W.C.W.).

Author information

Author notes

  1. Mark A Rossi, Dipendra K Aryal and Bence Racz: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Duke University Medical School, Durham, North Carolina, USA

    Il Hwan Kim, Akiyoshi Uezu, William C Wetsel & Scott H Soderling

  2. Department of Psychology and Neuroscience, Duke University Medical School, Durham, North Carolina, USA

    Mark A Rossi, Namsoo Kim & Henry Yin

  3. Department of Psychiatry and Behavioral Sciences, Duke University Medical School, Durham, North Carolina, USA

    Dipendra K Aryal & William C Wetsel

  4. Department of Psychiatry and Behavioral Sciences, Duke University Medical School, Durham, North Carolina

    Dipendra K Aryal & William C Wetsel

  5. Department of Anatomy and Histology, Szent István University, Budapest, Hungary

    Bence Racz

  6. Faculty of Veterinary Science, Szent István University, Budapest, Hungary

    Bence Racz

  7. Department of Neurobiology, Duke University Medical School, Durham, North Carolina, USA

    Fan Wang, William C Wetsel & Scott H Soderling

  8. Department of Cell Biology and Physiology, University of North Carolina, Chapel Hill, North Carolina, USA

    Richard J Weinberg

  9. Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, USA

    Richard J Weinberg

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Contributions

I.H.K. and S.H.S. designed this study. I.H.K. performed behavioral works, Golgi staining, cell biology, animal surgeries, virus infections, optical clearance of brains, immunohistochemistry, circuit tracings and imaging. I.H.K. and W.C.W. performed pharmacology studies. I.H.K. and A.U. performed three-dimensional reconstruction of dendritic segments. I.H.K., F.W. and S.H.S. performed virus design and purification. N.K. and H.Y. performed whole cell patch clamp recordings. D.K.A. and W.C.W. performed HPLC and microdialysis. M.A.R. and H.Y. performed optogenetics. B.R. and R.J.W. performed electron microscopy studies. I.H.K., D.K.A. and N.K. performed statistical analyses. This paper was written by I.H.K. and S.H.S. and was edited by the other authors.

Corresponding authors

Correspondence to Il Hwan Kim or Scott H Soderling.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Elevated vertical activity and stereotypical behavior in the Arp2/3 mutant mouse is normalized by antipsychotics, and re-plots of data from Figure 1 of haloperidol and clozapine in control mice.

(a) Open field analysis of rearing over time for Arp2/3 mutant (ArpC3f/f:CaMKIIαCre) or control (ArpC3f/f) mice given an intraperitoneal injection of vehicle (saline) or drug (haloperidol or clozapine) at 60min (arrow). (b) Cumulative vertical beam breaks per hour from (a) for each condition (*ps<0.05; two-way ANOVA with repeated measure followed by post-hoc tests). (c) Analysis of stereotypical activity (repeated beam breaks) in the open field over time for Arp2/3 mutant (ArpC3f/f:CaMKIIαCre) or control (ArpC3f/f) mice given (i.p.) of vehicle (saline) or drug (haloperidol or clozapine) at 60min (arrow). (d) Cumulative stereotypical activity per hour from (c) for each condition (*ps<0.05; two-way ANOVA with repeated measure followed by post-hoc tests). n=12-21. Each condition is color-coded according the key (top). Open field analysis of (e) distance traveled, (f) vertical activity, or (g) stereotypical activity over time for control (ArpC3f/f) mice given (i.p.) vehicle (saline) or drug (haloperidol or clozapine) at 60 min (dotted line). Each condition is color-coded according the key (top). @, #, and $ indicate statistical significances [@, vehicle vs. haloperidol (0.2mg/kg); #, vehicle vs. haloperidol (0.1mg/kg); $, vehicle vs. clozapine (0.5mg/kg)] in one way ANOVA with repeated measure followed by Bonferroni pair-wise comparisons. n=12-15. Data are presented as mean ±SEM. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 2 Camk2a-cre:Rosa26Tomato mouse (p42) shows Camk2a promoter–driven expression of Cre recombinase predominately in the cortex, with little expression in striatum, and acute haloperidol treatment does not rescue the spine density of Arp2/3 mutant mice.

(a) Schematic represents the regions of images in b-c of frontal cortex and striatum. (b-c) Representative images of Cre-dependent expression of RosaTomato (left panel), or DAPI stain (middle panel), and the merged images (right panel) from the frontal cortex (b) and the striatum (c). (d) Representative images of Golgi stained dendritic segments from the frontal cortical region (layers 3-5) of control (ArpC3f/f, top panel) (n=5), or Arp2/3 mutant mice (ArpC3f/f:CaMKIIαCre) treated with vehicle (saline, middle panel; n=5), or haloperidol (0.2 mg/kg, bottom panel; n=5) at p120-150. Vehicle and haloperidol (0.2mg/kg) was treated (i.p.) 1hr before perfusion. (e) Graph the average density of spines from (a) for each condition. Data are presented as mean ±SEM. N.S.; not statistically significant. All representative images were successfully repeated more than three times. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 3 Representative images depicting the Cre-dependent specific expression of the Flex-AAV viruses, and Cre-dependent expression of the Flex-AAV-ChR2 in HEK293T cells and mouse brain.

(a-b) Infection of Flex-AAV-GFP viruses do not express GFP in HEK293T cells transfected with tdTomato (row a), but do in the cells that express tdTomato-Cre (row b). (c-d) Infection of Flex-AAV-ArpC3-2A-GFP viruses do not express GFP in HEK293T cells transfected with tdTomato (row c), but do in the cells that express tdTomato-Cre (row d). (e) Infection of AAV-Flex-ChR2-mCherry expresses mCherry in GFP-Cre-positive HEK293 cells. Inset represents a high-magnification view showing membrane-associated expression of ChR2-mCherry. (f) In contrast, AAV-Flex-ChR2-mCherry does not express mCherry in the absence of Cre expression. (g-h) Frontal cortex of (g) CaMKII a-Cre mouse infected with AAV-Flex-ChR2-mCherry for two weeks results in the expression of mCherry, but the same infection into (h) WT frontal cortex does not result in mCherry expression. (i) Infection of AAV-Flex-ChR2-YFP virus expresses mCherry in GFP-Cre-positive HEK293 cells. Inset represents a high-magnification view showing a membranous expression of ChR2-YFP. (j) However, AAV-Flex-ChR2-YFP does not express YFP without Cre expression. (k-l) Frontal cortex of (k) CaMKII a-Cre mouse infected with AAV-Flex-ChR2-YFP for two weeks results in expression of YFP, but the same infection into (l) WT frontal cortex does not result in YFP expression. All representative images were successfully repeated more than three times.

Supplementary Figure 4 Flex-AAV-mediated regional rescue (FARR) of the frontal cortex in the Arp2/3 mutant mice normalizes rearing activity and stereotypical behavior, whereas FARR of the frontal cortex in the Arp2/3 mutant mice does not normalize PPI.

(a) Mean vertical beam breaks in the open field every 5 min for ArpC3f/f (gray circle; n=18), ArpC3f/f:CaMKIIαCre-GFP (cKO-control) (orange triangle; n=11), and ArpC3f/f:CaMKIIαCre-ArpC3-2A-GFP (cKO-rescue) (green diamond; n=15) mice. (b) Mean stereotypical activity (repeated beam breaks) in the open field every 5 min for ArpC3f/f (gray circle), ArpC3f/f:CaMKIIαCre-GFP (cKO-control) (orange triangle), and ArpC3f/f:CaMKIIαCre-ArpC3-2A-GFP (cKO-rescue) (green diamond) mice. (c) Percent prepulse inhibition (PPI) of the acoustic startle responses to the 4, 8, and 12 dB prepulses [n=16 for ArpC3f/f (WT); n=20 for ArpC3f/f:CaMKIIαCre-GFP (cKO-control); n=15 for ArpC3f/f:CaMKIIαCre-ArpC3-2A-GFP (cKO-rescue)]. One-way ANOVA followed by post-hoc tests reveal that WT mice show significantly higher PPI compared to both cKO groups. However, there were no statistical differences between cKO-control and cKO-rescue throughout all dB groups (4dB, 8dB, and 12 dB). *ps<0.05. Data are presented as mean ±SEM. N.S., not statistically significant. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 5 FLEX-AAV-mediated regional rescue (FARR) of the hippocampus in the Arp2/3 mutant mice does not normalize behavior in the open field.

(a) Schematic representation of the bilateral Cre-dependent ArpC3 rescue virus injection into hippocampus. (b) Representative serial coronal images showing ArpC3-2A-GFP (green) expression from dorsal to ventral hippocampus. (c to h) Analysis of open field behaviors following bilateral hippocampal rescue of Arp2/3. (c, e, g) Mean (c) distance traveled, (e) vertical activity, and (g) stereotypical behavior every 5 min for ArpC3f/f (WT) (gray line; n=14), ArpC3f/f:CaMKIIαCre-GFP (cKO-control) (orange line; n=14), and ArpC3f/f:CaMKIIαCre-ArpC3-2A-GFP (cKO-rescue) (green line n=12) mice. (d, f, h) Cumulative (d) distance traveled (f), vertical activity, and (h) stereotypical behavior for each group. Data are presented as mean ±SEM. N.S., not statistically significant. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 6 Frontal cortex–specific ArpC3 knockout leads to locomotor hyperactivity.

(a) Representative sagittal brain images showing the expressions of AAV-Synapsin I-GFP (top left) and AAV-CaMKII-Cre-GFP (top right) in frontal cortex (FC). Bottom panels showing the high-magnification views of top panels (red squares). Cre-GFP is predominantly expressed in the nucleus of frontal cortical cells (bottom right) compared to the cytoplasmic expression of GFP (bottom left). (b) Analysis of the locomotor activity of mice following bilateral infections of AAV-Synapsin I-GFP (control; gray line; n=16) or AAV-CaMKII-Cre-GFP (cKO; green line; n=14) in the frontal cortical region. One-way ANOVA with repeated measure followed by post-hoc tests reveal that Cre-GFP expressing mice show increased locomotor activity compared to GFP-expressing littermate controls from 25 to 60 min except at the 40 min time-point. *ps<0.05. Data are presented as mean ±SEM. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 7 Retrograde Cre expression by lenti-FuGB2-Cre virus, and ipsilateral projection of frontal cortex–VTA/SNc circuit.

(a) Schematic representation of the Lenti-FuGB2-Cre viral infection into the VTA/SNc of AI-14 Cre-reporter mouse. (b) Schematic illustration of tdTomato expression in a neuron which projects its axon into the VTA/SNc. (c) Infection of Lenti-FuGB2-Cre in VTA/SNc (white square) induces tdTomato expression in frontal cortical pyramidal neurons (yellow square) and striatal medium spiny neurons (sky blue square). Note that majority of tdTomato signals in VTA/SNc are cell body-free axonal fibers (white square). (d) Schematic representation of the circuit tracing based on a unilateral injection of Lenti-FuGB2-Cre virus into the VTA/SNc, followed by monitoring ipsilateral versus contralateral connectivity to the FC by bi-lateral injection of Flex-AAV-GFP. (e) Middle panel, image of a coronal section of the FC for GFP (green) and DAPI (blue). Boxed regions represent Left panel (ipsilateral image) and Right panel (contralateral image). All representative images were successfully repeated more than three times.

Supplementary Figure 8 Immunoelectron microscopy analyses of GABAergic synapses in the frontal cortex.

(a-b) Representative electron micrographs of GABAergic synapses (colored by blue) in the frontal cortical region of (a) ArpC3f/f and (b) ArpC3f/f:CaMKIIαCre mice. (c) Quantification of synapse density (n=43 micrographs from 3 mice for ArpC3f/f mice and n=46 micrographs from 3 mice for ArpC3f/f:CaMKIIαCre mice) (*p<0.001, independent t-test). Data are presented as mean ±SEM. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 9 cKO neurons display increased amplitude and frequency of mEPSCs in the frontal cortex.

Cumulative distribution of mEPSC (a) amplitude and (b) frequency. Each group [n=9 (10 DAI-WT; black line), n=10 (10 DAI-control; orange line), n=10 (10 DAI-rescue; blue line), n=15 (30 DAI-control; red line), n=15 (30 DAI-rescue; green line)] is color-coded according the key (center). DAI, days after infection. Detailed statistical analyses can be found in Supplementary Table 1.

Supplementary Figure 10 Schematic model proposing how loss of Arp2/3 activity leads to frontal cortical spine loss and increased locomotion via elevated dopamine in the striatum.

Progressive loss of spines downstream of Arp2/3 loss leads to abnormal synaptic contacts in the frontal cortex (FC; orange circle) (a), resulting in elevated frequency and amplitude of excitatory input of the KO neurons (b). This paradoxical excitation may enhance the activation of FC pyramidal neurons projecting to the VTA/SNc (c) that make synaptic contact with dopamine (DA) producing cells (blue circle) (d), leading to increased release of DA within the striatum (e). Finally, elevated levels of DA in the striatum provoke antipsychotic-responsive locomotor hyperactivity (f).

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Supplementary Figures 1–10 and Supplementary Table 1 (PDF 14315 kb)

Supplementary Methods Checklist (PDF 548 kb)

Pyramidal neurons of the frontal cortex project to and make synaptic contacts within the VTA/SNc.

Green signals indicate pyramidal neurons and their axons from the frontal cortex. Red signals show tyrosine hydroxylase–positive dopamine neurons in the VTA/SNc. Blue puncta indicate sites of Vglut1-positive excitatory synaptic contacts. (MOV 29560 kb)

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Kim, I., Rossi, M., Aryal, D. et al. Spine pruning drives antipsychotic-sensitive locomotion via circuit control of striatal dopamine. Nat Neurosci 18, 883–891 (2015). https://doi.org/10.1038/nn.4015

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