Skip to main content

Advertisement

SpringerLink
Log in

Molecular Profiling of VGluT1 AND VGluT2 Ventral Subiculum to Nucleus Accumbens Shell Projections

  • Original Paper
  • Published:
Neurochemical Research Aims and scope Submit manuscript

Abstract

The nucleus accumbens shell is a critical node in reward circuitry, encoding environments associated with reward. Long-range inputs from the ventral hippocampus (ventral subiculum) to the nucleus accumbens shell have been identified, yet their precise molecular phenotype remains to be determined. Here we used retrograde tracing to identify the ventral subiculum as the brain region with the densest glutamatergic (VGluT1–Slc17a7) input to the shell. We then used circuit-directed translating ribosome affinity purification to examine the molecular characteristics of distinct glutamatergic (VGluT1, VGluT2–Slc17a6) ventral subiculum to nucleus accumbens shell projections. We immunoprecipitated translating ribosomes from this population of projection neurons and analysed molecular connectomic information using RNA sequencing. We found differential gene enrichment across both glutamatergic projection neuron subtypes. In VGluT1 projections, we found enrichment of Pfkl, a gene involved in glucose metabolism. In VGluT2 projections, we found a depletion of Sparcl1 and Dlg1, genes known to play a role in depression- and addiction-related behaviours. These findings highlight potential glutamatergic neuronal-projection-specific differences in ventral subiculum to nucleus accumbens shell projections. Together these data advance our understanding of the phenotype of a defined brain circuit.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Data Availability

All data generated or analysed during this study are included in this published article. For additional information, please contact the corresponding authors.

References

  1. Salgado S, Kaplitt MG (2015) The nucleus accumbens: a comprehensive review. Stereotact Funct Neurosurg 93(2):75–93

    Article  PubMed  Google Scholar 

  2. Bossert JM et al (2007) Differential effects of blockade of dopamine D1-family receptors in nucleus accumbens core or shell on reinstatement of heroin seeking induced by contextual and discrete cues. J Neurosci 27(46):12655–12663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. French SJ, Totterdell S (2002) Hippocampal and prefrontal cortical inputs monosynaptically converge with individual projection neurons of the nucleus accumbens. J Comp Neurol 446(2):151–165

    Article  PubMed  Google Scholar 

  4. French S, Totterdell S (2003) Individual nucleus accumbens-projection neurons receive both basolateral amygdala and ventral subicular afferents in rats. Neuroscience 119(1):19–31

    Article  CAS  PubMed  Google Scholar 

  5. Böhm C et al (2018) Routes to, from and within the subiculum. Cell Tissue Res 373(3):557–563

    Article  PubMed  Google Scholar 

  6. Tsao A, Moser M-B, Moser EI (2013) Traces of experience in the lateral entorhinal cortex. Curr Biol 23(5):399–405

    Article  CAS  PubMed  Google Scholar 

  7. Marchant NJ et al (2016) Role of ventral subiculum in context-induced relapse to alcohol seeking after punishment-imposed abstinence. J Neurosci 36(11):3281–3294

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Bossert JM et al (2016) Role of projections from ventral subiculum to nucleus accumbens shell in context-induced reinstatement of heroin seeking in rats. Psychopharmacology 233(10):1991–2004

    Article  CAS  PubMed  Google Scholar 

  9. Anne C, Gasnier B (2014) Vesicular neurotransmitter transporters: mechanistic aspects. In: Current topics in membranes. Elsevier, pp 149–174

  10. El Mestikawy S et al (2011) From glutamate co-release to vesicular synergy: vesicular glutamate transporters. Nat Rev Neurosci 12(4):204–216

    Article  PubMed  Google Scholar 

  11. Shigeri Y, Seal RP, Shimamoto K (2004) Molecular pharmacology of glutamate transporters, EAATs and VGLUTs. Brain Res Brain Res Rev 45(3):250–265

    Article  CAS  PubMed  Google Scholar 

  12. Fremeau RT Jr et al (2002) The identification of vesicular glutamate transporter 3 suggests novel modes of signaling by glutamate. Proc Natl Acad Sci 99(22):14488–14493

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Schafer MK et al (2002) Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons. J Biol Chem 277(52):50734–50748

    Article  PubMed  Google Scholar 

  14. Gras C et al (2002) A third vesicular glutamate transporter expressed by cholinergic and serotoninergic neurons. J Neurosci 22(13):5442–5451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Herzog E et al (2004) Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain. Neuroscience 123(4):983–1002

    Article  CAS  PubMed  Google Scholar 

  16. Herzog E et al (2006) Synaptic and vesicular co-localization of the glutamate transporters VGLUT1 and VGLUT2 in the mouse hippocampus. J Neurochem 99(3):1011–1018

    Article  CAS  PubMed  Google Scholar 

  17. Fremeau RT Jr et al (2001) The expression of vesicular glutamate transporters defines two classes of excitatory synapse. Neuron 31(2):247–260

    Article  CAS  PubMed  Google Scholar 

  18. Varoqui H et al (2002) Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses. J Neurosci 22(1):142–155

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jin S et al (2022) Examining ventral subiculum and basolateral amygdala projections to the nucleus accumbens shell: Differential expression of VGLuT1, VGLuT2 and VGaT in the rat. Neurosci Lett 788:136858

    Article  CAS  PubMed  Google Scholar 

  20. Borgius L et al (2010) A transgenic mouse line for molecular genetic analysis of excitatory glutamatergic neurons. Mol Cell Neurosci 45(3):245–257

    Article  CAS  PubMed  Google Scholar 

  21. Paxinos G, Franklin KB (2004) The mouse brain in stereotaxic coordinates: compact. Elsevier, Amsterdam

    Google Scholar 

  22. Pazos A, Cortes R, Palacios JM (1985) Thyrotropin-releasing hormone receptor binding sites: autoradiographic distribution in the rat and guinea pig brain. J Neurochem 45:1448–1463

    Article  CAS  PubMed  Google Scholar 

  23. Nectow AR, Ekstrand MI, Friedman JM (2015) Molecular characterization of neuronal cell types based on patterns of projection with Retro-TRAP. Nat Protoc 10(9):1319–1327

    Article  CAS  PubMed  Google Scholar 

  24. Paxinos G, Franklin KB (2004) The mouse brain in stereotaxic coordinates. Gulf Professional Publishing

    Google Scholar 

  25. Heiman M et al (2014) Cell type-specific mRNA purification by translating ribosome affinity purification (TRAP). Nat Protoc 9(6):1282–1291

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ip CK et al (2019) Amygdala NPY circuits promote the development of accelerated obesity under chronic stress conditions. Cell Metab 30(1):111-128.e6

    Article  CAS  PubMed  Google Scholar 

  27. Liao Y, Shi W (2020) Read trimming is not required for mapping and quantification of RNA-seq reads at the gene level. NAR Genomics Bioinform 2(3):lqaa068

    Article  Google Scholar 

  28. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9(4):357–359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Liu Y, Schmidt B (2012) Long read alignment based on maximal exact match seeds. Bioinformatics 28(18):i318–i324

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kim D et al (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14(4):1–13

    Article  Google Scholar 

  31. Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31(2):166–169

    Article  CAS  PubMed  Google Scholar 

  32. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15(12):1–21

    Article  Google Scholar 

  33. Krishnan V et al (2007) Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 131(2):391–404

    Article  CAS  PubMed  Google Scholar 

  34. McCormack K et al (2002) Genetic analysis of the Mammalian K+ channel β subunit Kvβ2 (Kcnab2)∗. J Biol Chem 277(15):13219–13228

    Article  CAS  PubMed  Google Scholar 

  35. Peled-Kamar M et al (1998) Altered brain glucose metabolism in transgenic-PFKL mice with elevated L-phosphofructokinase: in vivo NMR studies. Brain Res 810(1–2):138–145

    Article  CAS  PubMed  Google Scholar 

  36. Block I et al (2019) CFP suppresses breast cancer cell growth by TES-mediated upregulation of the transcription factor DDIT3. Oncogene 38(23):4560–4573

    Article  CAS  PubMed  Google Scholar 

  37. Klingler A et al (2020) Species-, organ-and cell-type-dependent expression of SPARCL1 in human and mouse tissues. PLoS ONE 15(5):e0233422

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Nzounza P et al (2012) The scaffolding protein Dlg1 is a negative regulator of cell-free virus infectivity but not of cell-to-cell HIV-1 transmission in T cells. PLoS ONE 7(1):e30130

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Zhang Z et al (2011) Fuz regulates craniofacial development through tissue specific responses to signaling factors. PLoS ONE 6(9):e24608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Phillipson O, Griffiths A (1985) The topographic order of inputs to nucleus accumbens in the rat. Neuroscience 16(2):275–296

    Article  CAS  PubMed  Google Scholar 

  41. Groenewegen HJ et al (1999) Convergence and segregation of ventral striatal inputs and outputs. Ann N Y Acad Sci 877(1):49–63

    Article  CAS  PubMed  Google Scholar 

  42. Ekstrand MI et al (2014) Molecular profiling of neurons based on connectivity. Cell 157(5):1230–1242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Castellano P et al (2016) Methamphetamine compromises gap junctional communication in astrocytes and neurons. J Neurochem 137(4):561–575

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kokarovtseva L et al (2009) Excitability and gap junction–mediated mechanisms in nucleus accumbens regulate self-stimulation reward in rats. Neuroscience 159(4):1257–1263

    Article  CAS  PubMed  Google Scholar 

  45. Li C-Y, Mao X, Wei L (2008) Genes and (common) pathways underlying drug addiction. PLoS Comput Biol 4(1):e2

    Article  PubMed  PubMed Central  Google Scholar 

  46. Blendy JA, Maldonado R (1998) Genetic analysis of drug addiction: the role of cAMP response element binding protein. J Mol Med 76:104–110

    Article  CAS  PubMed  Google Scholar 

  47. Kohnhorst CL et al (2017) Identification of a multienzyme complex for glucose metabolism in living cells. J Biol Chem 292(22):9191–9203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Koponen H et al (2015) Association between suicidal behaviour and impaired glucose metabolism in depressive disorders. BMC Psychiatry 15(1):1–8

    Article  Google Scholar 

  49. Volkow ND et al (1994) Recovery of brain glucose metabolism in detoxified alcoholics: correction

  50. Kukurba KR, Montgomery SB (2015) RNA sequencing and analysis. Cold Spring Harbor Protoc 201(11):pdb-top084970

    Article  Google Scholar 

  51. Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Nat Rev Genet 12(2):87–98

    Article  CAS  PubMed  Google Scholar 

  52. Hindson CM et al (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Methods 10(10):1003–1005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Harris MJ, Juriloff DM (2010) An update to the list of mouse mutants with neural tube closure defects and advances toward a complete genetic perspective of neural tube closure. Birth Defects Res A 88(8):653–669

    Article  CAS  Google Scholar 

  54. Ayimugu A et al (2020) Investigation of the involvement of Parkin in Parkinson’s disease and cancer by monitoring the changes in SH-SY5Y cells at the nuclear proteome level. Anticancer Res 40(6):3169–3190

    Article  CAS  PubMed  Google Scholar 

  55. Irie Y et al (2011) Methamphetamine induces endoplasmic reticulum stress related gene CHOP/Gadd153/ddit3 in dopaminergic cells. Cell Tissue Res 345(2):231–241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Maiya R et al (2012) DlgS97/SAP97, a neuronal isoform of discs large, regulates ethanol tolerance. PLoS ONE 7(11):e48967

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Vialou V et al (2010) ΔFosB in brain reward circuits mediates resilience to stress and antidepressant responses. Nat Neurosci 13(6):745–752

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Hindson BJ et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83(22):8604–8610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Wang S, Hodges S, Skvortsow D (2013) Droplet digital PCR: ultra-sensitive validation technology for RNA-seq

Download references

Acknowledgements

This project involved several multi-disciplinary and cross-institutional collaborations. We would firstly like to thank Dr. Alexander Nectow and Professor Jeffrey Friedman from The Rockefeller University for providing the pAAV-EF1a-FLEX-NBL10-HA construct and critical methodological information. We acknowledge The Rockefeller University Bi-Institutional Antibody & Bioresource Core Facility at the Memorial Sloan Kettering Cancer Center for providing the Htz GFP monoclonal antibodies for immunoprecipitation. We thank Professor Zane Andrews at Monash University for providing VGluT1- and VGluT2-cre mice. We acknowledge the assistance of Dr. Vicky Perreau, Professor Christine Wells, Samuel Tanner and Franca Casagranda from The University of Melbourne for assistance with initial experimental design, bioinformatic analysis and ddPCR probe design. We thank Kym Pham from The University of Melbourne Center for Cancer Research for assistance with RNA sequencing. Finally, we thank Dr. Fatemeh Zanganeh, Dr. Sarah Ch’ng, Dr. Leigh Walker, Dr. Roberta Anversa, Associate Professor Daniel Scott and Liubov Lee-Kardashyan from the Florey Institute for assistance with experimental procedures, bioinformatic analysis and constructive conceptual input.

Funding

This work was supported by a National Health and Medical Research Council Project Grant to AJL and EJC (1159032) and a National Health and Medical Research Council Synergy Grant to AJL (2009851), a Brain and Behavior Research Foundation NARSAD Young Investigator Grant to EJC (28007) and a University of Melbourne Early Career Researcher Grant to EJC. We acknowledge the Victorian State Government Operational Infrastructure Scheme. This work was also supported in part through the NIH/NCI Cancer Center Support Grant GrantP30 CA008748 which funds The Rockefeller University Bi-Institutional Antibody and Bioresource Core Facility.

Author information

Author notes

  1. Shubo Jin and Erin J. Campbell: Co-first authors.

Authors and Affiliations

  1. The Florey Institute of Neuroscience and Mental Health, Melbourne Brain Centre, The University of Melbourne, Parkville, Melbourne, VIC, 3052, Australia

    Shubo Jin, Erin J. Campbell, Sharon Layfield, Ross A. D. Bathgate & Andrew J. Lawrence

  2. Florey Department of Neuroscience and Mental Health, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia

    Shubo Jin, Erin J. Campbell, Sharon Layfield, Ross A. D. Bathgate & Andrew J. Lawrence

  3. Department of Biochemistry and Pharmacology, The University of Melbourne, Parkville, Melbourne, VIC, 3010, Australia

    Ross A. D. Bathgate

  4. Neuroscience Division, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, 2010, Australia

    Chi Kin Ip & Herbert Herzog

  5. Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia

    Chi Kin Ip & Herbert Herzog

Authors
  1. Shubo Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Erin J. Campbell
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chi Kin Ip
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sharon Layfield
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ross A. D. Bathgate
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Herbert Herzog
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andrew J. Lawrence
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Study conception by AJL. All authors contributed to the study design. Material preparation, data collection and analysis were performed by SJ, EJC, CKI and SL. The first draft of the manuscript was written by EJC, SJ and AJL. All authors edited and approved the final manuscript.

Corresponding authors

Correspondence to Erin J. Campbell or Andrew J. Lawrence.

Ethics declarations

Competing Interests

All authors report no competing interests to disclose.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 23 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jin, S., Campbell, E.J., Ip, C.K. et al. Molecular Profiling of VGluT1 AND VGluT2 Ventral Subiculum to Nucleus Accumbens Shell Projections. Neurochem Res 48, 2490–2501 (2023). https://doi.org/10.1007/s11064-023-03921-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11064-023-03921-z

Keywords

Search

Navigation