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VTA CRF neurons mediate the aversive effects of nicotine withdrawal and promote intake escalation

Nature Neuroscience volume 17, pages 1751–1758 (2014)Cite this article

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Abstract

Dopaminergic neurons in the ventral tegmental area (VTA) are well known for mediating the positive reinforcing effects of drugs of abuse. Here we identify in rodents and humans a population of VTA dopaminergic neurons expressing corticotropin-releasing factor (CRF). We provide further evidence in rodents that chronic nicotine exposure upregulates Crh mRNA (encoding CRF) in dopaminergic neurons of the posterior VTA, activates local CRF1 receptors and blocks nicotine-induced activation of transient GABAergic input to dopaminergic neurons. Local downregulation of Crh mRNA and specific pharmacological blockade of CRF1 receptors in the VTA reversed the effect of nicotine on GABAergic input to dopaminergic neurons, prevented the aversive effects of nicotine withdrawal and limited the escalation of nicotine intake. These results link the brain reward and stress systems in the same brain region to signaling of the negative motivational effects of nicotine withdrawal.

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Figure 1: Nicotine dependence increases Crh mRNA in the VTA.
Figure 2: Identification of VTA CRF neurons in mice.
Figure 3: Double labeling of CRF/DA neurons using Crh in situ hybridization and TH immunohistochemistry in mice.
Figure 4: Double labeling of CRF/DA neurons using CRH in situ hybridization and TH immunohistochemistry in humans.
Figure 5: Nicotine dependence causes dysregulation of GABA-DA synapses, which is reversed by downregulation of Crh mRNA.
Figure 6: Viral vector–mediated downregulation of Crh mRNA in the VTA prevents the aversive motivational response to nicotine withdrawal.
Figure 7: Viral vector-mediated downregulation of Crh mRNA in the VTA decreases long-access nicotine self-administration and prevents abstinence-induced escalation of nicotine intake.
Figure 8: CRF1 receptor antagonism prevents the aversive motivational response to withdrawal from chronic nicotine.

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Acknowledgements

The authors thank M. Brennan, B. Takabe, C. Arias and the University of Toronto Division of Comparative Medicine staff for technical assistance and M. Arends for editorial assistance. The authors would also like to thank R. Nagra and J. Riehl and the UCLA Brain Bank (The Human Brain and Spinal Fluid Resource Center) for providing the human samples. This work was supported by the Canadian Institutes of Health Research, US National Institute on Drug Abuse (DA023597, DA035371 and DA031566), US National Institute on Alcohol Abuse and Alcoholism (AA021491, AA015566, F32 AA020430, AA006420, AA016658, AA021667 and INIA AA013498), Tobacco-Related Disease Research Program (12RT-0099), US National Institute of Diabetes and Digestive and Kidney Diseases (DK026741) and the Clayton Medical Research Foundation.

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Authors and Affiliations

  1. Institute of Medical Science and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada

    Taryn E Grieder, Hector Vargas-Perez, Michal Chwalek, Geith Maal-Bared, Laura Clarke & Derek van der Kooy

  2. Committee on the Neurobiology of Addictive Disorders, The Scripps Research Institute, La Jolla, California, USA

    Melissa A Herman, Candice Contet, Ami Cohen, John Freiling, Joel E Schlosburg, Elena Crawford, Marisa Roberto & Olivier George

  3. The Salk Institute, La Jolla, California, USA

    Laura A Tan & Paul E Sawchenko

  4. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS / INSERM / Université de Strasbourg, Illkirch, France

    Pascale Koebel & Brigitte L Kieffer

  5. Molecular and Cellular Neuroscience, The Scripps Research Institute, La Jolla, California, USA

    Vez Repunte-Canonigo & Pietro P Sanna

  6. Brudnick Neuropsychiatric Research Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Andrew R Tapper

  7. Department of Psychiatry, Douglas Hospital Research Center, McGill University, Montreal, Quebec, Canada

    Brigitte L Kieffer

  8. National Institute on Alcohol Abuse and Alcoholism, Rockville, Maryland, USA

    George F Koob

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  1. Taryn E Grieder
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Contributions

T.E.G. and O.G. designed the experiments. T.E.G., H.V.-P., M.C., J.E.S. and G.M.-B. performed minipump, cannulation and viral vector surgeries. M.R. and M.A.H. performed the electrophysiology experiments. T.E.G. performed place conditioning and open field testing. A.C. performed self-administration experiments. C.C., L.A.T. and P.E.S. performed ISH. C.C. and E.C. performed double ISH and immunohistochemistry. T.E.G., V.R.-C., P.P.S., A.R.T. and L.C. performed molecular studies. J.F. and E.C. performed immunohistochemistry. C.C., P.K. and B.L.K. supplied viral vectors. T.E.G. and O.G. analyzed the data. T.E.G., C.C., G.F.K., D.v.d.K. and O.G. wrote the paper. All of the authors discussed the results and read the paper.

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Correspondence to Olivier George.

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Integrated supplementary information

Supplementary Figure 1 Crh mRNA relative expression in the CeA, VTA and PVN.

Crh mRNA levels in the CeA, VTA, and PVN. Crh mRNA levels in the CeA, VTA, and PVN relative to the housekeeping gene GAPDH in mice chronically exposed to saline, measured by RT-PCR (n = 13 or 14 per group). Lower levels of Crh mRNA were observed in the VTA compared with the CeA and PVN (*p < 0.05).

Supplementary Figure 2 Number of Crh mRNA–positive neurons in the VTA

Number of CRF neurons per section in the VTA (bregma range: -2.92 to -3.88) in saline minipump, saline minipump + acute nicotine (1.5 mg/kg), nicotine minipump (12 days), and nicotine minipump and withdrawn (8 h) groups of mice (n = 6 per group). No difference was observed between groups. Data in Fig. 2 in the manuscript represents combined data from the four groups.

Supplementary Figure 3 Urocortin in situ hybridization in the VTA and Edinger-Westphal nucleus.

Notice the lack of urocortin neurons in the VTA and the prominent population of urocortin-positive neurons in the Edinger-Westphal nucleus. This result was repeated on 6 naïve and 6 nicotine dependent mice. Urocortin neurons were never observed in the VTA.

Supplementary Figure 4 CRF immunodensity in the VTA, CeA, PVN and IPN in nicotine-dependent and withdrawn mice.

Quantification of immunodensity in the aVTA and pVTA (a), PVN (b), CeA (c) and IPN (d) in nondependent mice treated with saline (Sal), nicotine-dependent mice (Nic) and nicotine-dependent and -withdrawn mice (WD; n = 12-26 mice per group). e. Example of CRF-immunostained neuropils in the CeA showing CRF varicosities in a transversal fiber. Densitometry analysis revealed that both nicotine dependence and withdrawal from chronic nicotine decreased CRF peptide density compared with saline-treated mice in the pVTA (F2,31 = 4.40, p = 0.02; Fig. S4a), interpeduncular nucleus (IPN: F2,49 = 4.24, p = 0.020; Fig S4d), and CeA (F2,67 = 3.15, p = 0.049; Fig. S4c) but not aVTA (F2,23 = 0.03, p = 0.97; Fig. S4a) or PVN (F2,21 = 0.75, p = 0.48; Fig. S4b). Note that a different number of sections were used for the different brain regions resulting in a different degree of freedom. This experiment was performed with 1 cohort.

Supplementary Figure 5 Validation of the AAV2-shCRH viral vector.

a, Timeline of viral vector infusion and behavioral experiments in mice. b, DNA construct used to produce vectors for CRF silencing (AAV2-shCRH) and control vectors (AAV2-shSCR). ITR, inverted terminal repeat; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; hGH polyA, human growth hormone polyadenylation signal. c, Representative images of VTA Crh mRNA-containing cells taken at 20× magnification. d, Number of Crh mRNA-containing cells per section. Quantification of Crh-positive neurons restricted to GFP-positive areas in the pVTA revealed significant downregulation (-26%) of the number of Crh mRNA-containing cells in AAV2-shCRH mice compared with AAV2-shSCR mice (t12 = 2.7, p = 0.020). AAV2-shCRH also decreased the total number of CRF neurons in the entire VTA (including the region without GFP) by 18% (t12 = 2.4, p = 0.033). Notably, the mice were sacrificed approximately 4 weeks into withdrawal after the end of behavioral testing, demonstrating that the upregulation of Crh mRNA in nicotine-withdrawn mice and CRF silencing by the viral vector were both long-lasting. This experiment was performed with 1 cohort.

Supplementary Figure 6 Electrophysiological recording of VTA DA neurons.

a, Representative trace of a recorded VTA DA neuron. b, Definition of DA neuron action potential characteristics. c, Characterization of VTA dopamine neurons from naive (n = 8 cells), nicotine-dependent AAV2-shSCR (n = 11 cells), and nicotine-dependent AAV2-shCRH (n = 12 cells) mice (n = 3 naïve, n = 4 nicotine-dependent AAV2-shSCR, n = 4 nicotine-dependent AAV2-shCRH). This experiment was performed with 1 cohort.

Supplementary Figure 7 Comparison of confirmed TH-positive VTA cells with all cells examined.

a, Effects of acute nicotine in naive mice on sIPSC frequency in all VTA cells examined (left; *p = 0.0015 by paired t-test) compared with TH+-only VTA cells (right; *p = 0.0051). The total number of cells for each condition is indicated in parentheses. b, Average change in sIPSC frequency in all VTA cells examined (left; *p = 0.0026, one-sample t-test; #p = 0.0003, unpaired t-test) compared with TH+-only VTA cells (right; *p = 0.0128, one-sample t-test; #p = 0.0005, unpaired t-test). Exact t and p values noted under each condition. Total number of cells for each condition indicated in parentheses.

Supplementary Figure 8 Anatomical specificity of AAV2-shCRH VTA injections.

Mice with AAV2-shCRH injections outside of the VTA (n = 7) showed an aversive motivational response to nicotine withdrawal that was not significantly different from mice injected with AAV2-shSCR (n = 11).

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Grieder, T., Herman, M., Contet, C. et al. VTA CRF neurons mediate the aversive effects of nicotine withdrawal and promote intake escalation. Nat Neurosci 17, 1751–1758 (2014). https://doi.org/10.1038/nn.3872

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