Dopamine (DA) signaling is critical for movement, motivation, and addictive behavior. The neuronal GTPase, Rit2, is enriched in DA neurons (DANs), binds directly to the DA transporter (DAT), and is implicated in several DA-related neuropsychiatric disorders. However, it remains unknown whether Rit2 plays a role in either DAergic signaling and/or DA-dependent behaviors. Here we leveraged the TET-OFF system to conditionally silence Rit2 in Pitx3IRES2-tTA mouse DANs. Following DAergic Rit2 knockdown (Rit2-KD), mice displayed an anxiolytic phenotype, with no change in baseline locomotion. Further, males exhibited increased acute cocaine sensitivity, whereas DAergic Rit2-KD suppressed acute cocaine sensitivity in females. DAergic Rit2-KD did not affect presynaptic TH and DAT protein levels in females, nor was TH was affected in males; however, DAT was significantly diminished in males. Paradoxically, despite decreased DAT levels in males, striatal DA uptake was enhanced, but was not due to enhanced DAT surface expression in either dorsal or ventral striatum. Finally, patch recordings in nucleus accumbens (NAcc) medium spiny neurons (MSNs) revealed reciprocal changes in spontaneous EPSP (sEPSP) frequency in male and female D1+ and D2+ MSNs following DAergic Rit2-KD. In males, sEPSP frequency was decreased in D1+, but not D2+, MSNs, whereas in females sEPSP frequency decreased in D2+, but not D1+, MSNs. Moreover, DAergic Rit2-KD abolished the ability of cocaine to reduce sEPSP frequency in D1+, but not D2+, male MSNs. Taken together, our studies are among the first to acheive AAV-mediated, conditional and inducible DAergic knockdown in vivo. Importantly, our results provide the first evidence that DAergic Rit2 expression differentially impacts striatal function and DA-dependent behaviors in males and females.
Subscribe to Journal
Get full journal access for 1 year
only $38.38 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Wise RA. Dopamine, learning and motivation. Nat Rev Neurosci. 2004;5:483–94.
Russo SJ, Nestler EJ. The brain reward circuitry in mood disorders. Nat Rev Neurosci. 2013;14:609–25.
Calhoon GG, Tye KM. Resolving the neural circuits of anxiety. Nat Neurosci. 2015;18:1394–404.
Poewe W, Seppi K, Tanner CM, Halliday GM, Brundin P, Volkmann J, et al. Parkinson disease. Nat Rev Dis Prim. 2017;3:17013.
Faraone SV, Asherson P, Banaschewski T, Biederman J, Buitelaar JK, Ramos-Quiroga JA, et al. Attention-deficit/hyperactivity disorder. Nat Rev Dis Prim. 2015;1:15020.
Grace AA. Dysregulation of the dopamine system in the pathophysiology of schizophrenia and depression. Nat Rev Neurosci. 2016;17:524–32.
Kristensen AS, Andersen J, Jorgensen TN, Sorensen L, Eriksen J, Loland CJ, et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev. 2011;63:585–640.
Zhou Q, Li J, Wang H, Yin Y, Zhou J. Identification of nigral dopaminergic neuron-enriched genes in adult rats. Neurobiol Aging. 2011;32:313–26.
Spencer ML, Shao H, Tucker HM, Andres DA. Nerve growth factor-dependent activation of the small GTPase Rin. J Biol Chem. 2002;277:17605–15.
Shi GX, Han J, Andres DA. Rin GTPase couples nerve growth factor signaling to p38 and b-Raf/ERK pathways to promote neuronal differentiation. J Biol Chem. 2005;280:37599–609.
Uenaka T, Satake W, Cha PC, Hayakawa H, Baba K, Jiang S, et al. In silico drug screening by using genome-wide association study data repurposed dabrafenib, an anti-melanoma drug, for Parkinson’s disease. Hum Mol Genet. 2018;27:3974–85.
Hamedani SY, Gharesouran J, Noroozi R, Sayad A, Omrani MD, Mir A, et al. Ras-like without CAAX 2 (RIT2): a susceptibility gene for autism spectrum disorder. Metab Brain Dis. 2017;32:751–55.
Daneshmandpour Y, Darvish H, Emamalizadeh B. RIT2: responsible and susceptible gene for neurological and psychiatric disorders. Mol Genet Genom. 2018;293:785–92.
Emamalizadeh B, Jamshidi J, Movafagh A, Ohadi M, Khaniani MS, Kazeminasab S, et al. RIT2 Polymorphisms: Is There a Differential Association? Mol Neurobiol. 2017;54:2234–40.
Glessner JT, Reilly MP, Kim CE, Takahashi N, Albano A, Hou C, et al. Strong synaptic transmission impact by copy number variations in schizophrenia. Proc Natl Acad Sci USA. 2010;107:10584–9.
Navaroli DM, Stevens ZH, Uzelac Z, Gabriel L, King MJ, Lifshitz LM, et al. The plasma membrane-associated GTPase Rin interacts with the dopamine transporter and is required for protein kinase C-regulated dopamine transporter trafficking. J Neurosci. 2011;31:13758–70.
Xie J, Mao Q, Tai PWL, He R, Ai J, Su Q, et al. Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity. Mol Ther. 2017;25:1363–74.
Wu Z, Yang H, Colosi P. Effect of genome size on AAV vector packaging. Mol Ther. 2010;18:80–6.
Gabriel LR, Wu S, Kearney P, Bellve KD, Standley C, Fogarty KE, et al. Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton. J Neurosci. 2013;33:17836–46.
Wu S, Bellve KD, Fogarty KE, Melikian HE. Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant. Proc Natl Acad Sci USA. 2015;112:15480–5.
Lin X, Parisiadou L, Sgobio C, Liu G, Yu J, Sun L, et al. Conditional expression of Parkinson’s disease-related mutant alpha-synuclein in the midbrain dopaminergic neurons causes progressive neurodegeneration and degradation of transcription factor nuclear receptor related 1. J Neurosci. 2012;32:9248–64.
Beninger RJ. The role of dopamine in locomotor activity and learning. Brain Res. 1983;287:173–96.
Gunaydin LA, Grosenick L, Finkelstein JC, Kauvar IV, Fenno LE, Adhikari A, et al. Natural neural projection dynamics underlying social behavior. Cell. 2014;157:1535–51.
Swapna I, Bondy B, Morikawa H. Differential dopamine regulation of Ca(2+) signaling and its timing dependence in the nucleus accumbens. Cell Rep 2016;15:563–73.
Nicola SM, Malenka RC. Dopamine depresses excitatory and inhibitory synaptic transmission by distinct mechanisms in the nucleus accumbens. J Neurosci. 1997;17:5697–710.
Cepeda C, Buchwald NA, Levine MS. Neuromodulatory actions of dopamine in the neostriatum are dependent upon the excitatory amino acid receptor subtypes activated. Proc Natl Acad Sci USA. 1993;90:9576–80.
Nicola SM, Malenka RC. Modulation of synaptic transmission by dopamine and norepinephrine in ventral but not dorsal striatum. J Neurophysiol. 1998;79:1768–76.
Marshall JF, Berrios N. Movement disorders of aged rats: reversal by dopamine receptor stimulation. Science. 1979;206:477–9.
Starr BS, Starr MS. Differential effects of dopamine D1 and D2 agonists and antagonists on velocity of movement, rearing and grooming in the mouse. Implications for the roles of D1 and D2 receptors. Neuropharmacology. 1986;25:455–63.
Lotharius J, Brundin P. Pathogenesis of Parkinson’s disease: dopamine, vesicles and alpha-synuclein. Nat Rev Neurosci. 2002;3:932–42.
Howes O, McCutcheon R, Stone J. Glutamate and dopamine in schizophrenia: an update for the 21st century. J Psychopharmacol. 2015;29:97–115.
Volkow ND, Wise RA, Baler R. The dopamine motive system: implications for drug and food addiction. Nat Rev Neurosci. 2017;18:741–52.
Aschauer DF, Kreuz S, Rumpel S. Analysis of transduction efficiency, tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS ONE. 2013;8:e76310.
Korotkova TM, Ponomarenko AA, Haas HL, Sergeeva OA. Differential expression of the homeobox gene Pitx3 in midbrain dopaminergic neurons. Eur J Neurosci. 2005;22:1287–93.
Smidt MP, von Oerthel L, Hoekstra EJ, Schellevis RD, Hoekman MF. Spatial and temporal lineage analysis of a Pitx3-driven Cre-recombinase knock-in mouse model. PLoS ONE. 2012;7:e42641.
Tillack K, Aboutalebi H, Kramer ER. An Efficient and Versatile System for Visualization and Genetic Modification of Dopaminergic Neurons in Transgenic Mice. PLoS ONE. 2015;10:e0136203.
Cagniard B, Beeler JA, Britt JP, McGehee DS, Marinelli M, Zhuang X. Dopamine scales performance in the absence of new learning. Neuron. 2006;51:541–7.
Lee CH, Della NG, Chew CE, Zack DJ. Rin, a neuron-specific and calmodulin-binding small G-protein, and Rit define a novel subfamily of ras proteins. J Neurosci. 1996;16:6784–94.
Pannell M, Cai W, Brelsfoard J, Carlson S, Littlejohn E, Stewart T, et al. Rin GTPase deficiency promotes neuroprotection following traumatic brain injury. FASEB J. 2015;29(1_supplement):727.15.
Zweifel LS, Fadok JP, Argilli E, Garelick MG, Jones GL, Dickerson TM, et al. Activation of dopamine neurons is critical for aversive conditioning and prevention of generalized anxiety. Nat Neurosci. 2011;14:620–6.
Bahi A, Dreyer J-L. Dopamine transporter (DAT) knockdown in the nucleus accumbens improves anxiety- and depression-related behaviors in adult mice. Behav Brain Res. 2019;359:104–15.
Burke DA, Rotstein HG, Alvarez VA. Striatal local circuitry: a new framework for lateral inhibition. Neuron. 2017;96:267–84.
Silm K, Yang J, Marcott PF, Asensio CS, Eriksen J, Guthrie DA, et al. Synaptic vesicle recycling pathway determines neurotransmitter content and release properties. Neuron. 2019;102(4):786–800.e5.
Tilley MR, Cagniard B, Zhuang X, Han DD, Tiao N, Gu HH. Cocaine reward and locomotion stimulation in mice with reduced dopamine transporter expression. BMC Neurosci. 2007;8:42.
Nelson AM, Larson GA, Zahniser NR. Low or high cocaine responding rats differ in striatal extracellular dopamine levels and dopamine transporter number. J Pharm Exp Ther. 2009;331:985–97.
Becker JB, Koob GF. Sex differences in animal models: focus on addiction. Pharmacol Rev. 2016;68:242–63.
Calipari ES, Juarez B, Morel C, Walker DM, Cahill ME, Ribeiro E, et al. Dopaminergic dynamics underlying sex-specific cocaine reward. Nat Commun. 2017;8:13877.
Renthal W, Kumar A, Xiao G, Wilkinson M, Covington HE 3rd, Maze I, et al. Genome-wide analysis of chromatin regulation by cocaine reveals a role for sirtuins. Neuron. 2009;62:335–48.
Lobo MK, Covington HE 3rd, Chaudhury D, Friedman AK, Sun H, Damez-Werno D, et al. Cell type-specific loss of BDNF signaling mimics optogenetic control of cocaine reward. Science. 2010;330:385–90.
Kravitz AV, Tye LD, Kreitzer AC. Distinct roles for direct and indirect pathway striatal neurons in reinforcement. Nat Neurosci. 2012;15:816–8.
Kupchik YM, Brown RM, Heinsbroek JA, Lobo MK, Schwartz DJ, Kalivas PW. Coding the direct/indirect pathways by D1 and D2 receptors is not valid for accumbens projections. Nat Neurosci. 2015;18:1230–2.
Soares-Cunha C, Coimbra B, David-Pereira A, Borges S, Pinto L, Costa P, et al. Activation of D2 dopamine receptor-expressing neurons in the nucleus accumbens increases motivation. Nat Commun. 2016;7:11829.
Nicola SM, Kombian SB, Malenka RC. Psychostimulants depress excitatory synaptic transmission in the nucleus accumbens via presynaptic D1-like dopamine receptors. J Neurosci. 1996;16:1591–604.
Trantham-Davidson H, Lavin A. Acute cocaine administration depresses cortical activity. Neuropsychopharmacology. 2004;29:2046–51.
Wu N, Cepeda C, Zhuang X, Levine MS. Altered corticostriatal neurotransmission and modulation in dopamine transporter knock-down mice. J Neurophysiol. 2007;98:423–32.
Gerfen CR, Surmeier DJ. Modulation of striatal projection systems by dopamine. Annu Rev Neurosci. 2011;34:441–66.
Chen R, Daining CP, Sun H, Fraser R, Stokes SL, Leitges M, et al. Protein kinase Cbeta is a modulator of the dopamine D2 autoreceptor-activated trafficking of the dopamine transporter. J Neurochem. 2013;125:663–72.
Gowrishankar R, Gresch PJ, Davis GL, Katamish RM, Riele JR, Stewart AM, et al. Region-specific regulation of presynaptic dopamine homeostasis by d2 autoreceptors shapes the in vivo impact of the neuropsychiatric disease-associated DAT variant Val559. J Neurosci. 2018;38:5302–12.
We would like to acknowledge Dr. Susanna Casacuberta for outstanding surgical training, and Tucker Conklin for excellent technical assistance.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Sweeney, C.G., Kearney, P.J., Fagan, R.R. et al. Conditional, inducible gene silencing in dopamine neurons reveals a sex-specific role for Rit2 GTPase in acute cocaine response and striatal function. Neuropsychopharmacol. 45, 384–393 (2020). https://doi.org/10.1038/s41386-019-0457-x
Neurochemical Research (2020)
Journal of Biological Chemistry (2020)
Journal of Neurochemistry (2020)
Neurochemical Research (2020)