Skip to main content

Inhibitory synaptic transmissions to the bed nucleus of the stria terminalis neurons projecting to the ventral tegmental area are enhanced in rats exposed to chronic mild stress

Abstract

The comorbidities of depression and chronic pain have long been recognized in the clinic, and several preclinical studies have demonstrated depression-like behaviors in animal models of chronic pain. These findings suggest a common neuronal basis for depression and chronic pain. Recently, we reported that the mesolimbic dopaminergic system was tonically suppressed during chronic pain by enhanced inhibitory synaptic inputs to neurons projecting from the dorsolateral bed nucleus of the stria terminalis (dlBNST) to the ventral tegmental area (VTA), suggesting that tonic suppression of the mesolimbic dopaminergic system by this neuroplastic change may be involved in chronic pain-induced depression-like behaviors. In this study, we hypothesized that inhibitory synaptic inputs to VTA-projecting dlBNST neurons are also enhanced in animal models of depression, thereby suppressing the mesolimbic dopaminergic system. To test this hypothesis, we performed whole-cell patch-clamp electrophysiology using brain slices prepared from rats exposed to chronic mild stress (CMS), a widely used animal model of depression. The results showed a significant enhancement in the frequency of spontaneous inhibitory postsynaptic currents in VTA-projecting dlBNST neurons in the CMS group compared with the no stress group. The findings revealed enhanced inhibitory synaptic inputs to VTA-projecting dlBNST neurons in this rat model of depression, suggesting that this neuroplastic change is a neuronal mechanism common to depression and chronic pain that causes dysfunction of the mesolimbic dopaminergic system, thereby inducing depression-like behaviors.

Introduction

The comorbidities of depression and chronic pain have long been recognized in the clinic [1], and several preclinical studies have shown depression-like behaviors in animal models of chronic pain [2]. These findings suggest a common neuronal basis for depression and chronic pain. The mesolimbic dopaminergic pathway from the ventral tegmental area (VTA) to the nucleus accumbens (NAc) is a key player in the mesolimbic reward circuit, and dysfunction in this pathway has been implicated in depression [3]. We previously reported that reward-induced dopamine release in the NAc was suppressed in rats exposed to chronic mild stress (CMS) model [4], a widely used animal model of depression [5]. Dysfunction of the mesolimbic dopaminergic pathway has also been implicated in chronic pain. We demonstrated that reward-induced dopamine release in the NAc [6] and reward-seeking behaviors [7] were suppressed in rats with chronic pain. These findings suggest that suppression of the mesolimbic reward circuit may be a common neuroplastic change underlying depression and chronic pain. Recently, we showed that the mesolimbic dopaminergic system was tonically suppressed during chronic pain by enhanced inhibitory synaptic inputs to neurons projecting from the dorsolateral bed nucleus of the stria terminalis (dlBNST) to the VTA [8]. We previously reported that most VTA-projecting BNST neurons are GABAergic neurons that preferentially form synapses on VTA GABAergic neurons [9]. Thus, enhanced inhibitory synaptic inputs to VTA-projecting dlBNST neurons should activate VTA GABAergic neurons via a disinhibition mechanism, thereby causing suppression of VTA dopaminergic neurons that may induce depression and anhedonia observed in animal models of chronic pain. In the present study, we hypothesized that inhibitory synaptic inputs to VTA-projecting dlBNST neurons are also enhanced in animal models of depression, thereby suppressing the mesolimbic dopaminergic system. To test this hypothesis, we investigated inhibitory inputs to VTA-projecting dlBNST neurons by whole-cell patch-clamp electrophysiology using brain slices prepared from rats exposed to CMS.

Materials and methods

Male Sprague-Dawley rats (4 weeks old at the start of stress exposure) were exposed to the eight types of stress shown in Fig. 1a (CMS group) or no stress (NS group) over a 4-week period, according to the schedule shown in Fig. 1b. Electrophysiological experiments were performed within 1 week after the final stress exposure. To visualize VTA-projecting dlBNST neurons, red retrobeads were injected into the VTA (5.5 mm rostral, 1.0 mm lateral, 9.0 mm ventral to bregma) 3–7 days before the electrophysiological experiments (Fig. 1c). The electrophysiological experiments were performed as described previously [8]. Briefly, rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with ice-cold cutting solution. Their brains were quickly removed, and coronal slices (250 µm thick) containing the BNST were prepared in ice-cold cutting solution using a vibratome. The slices were transferred to a submerged recording chamber on an upright microscope and continuously superfused with recording solution at 35 ± 1 °C, saturated with 95% O2/5% CO2, at a flow rate of 1 ml/min. VTA-projecting dlBNST neurons labeled with retrobeads were visualized using epifluorescence and initially classified into the three types of neurons in current-clamp mode. We previously reported that approximately 80% of VTA-projecting dlBNST neurons are type III [10]. Thus, in this study, spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded from VTA-projecting dlBNST type III neurons, in the presence of 2 mM kynurenic acid to inhibit excitatory postsynaptic currents. In the experiments to examine the effect of NBI27914, a selective corticotropin-releasing factor (CRF) type I receptor antagonist, on sIPSCs, 1 µM NBI27914 was perfused for 15 min, and sIPSCs were analyzed during the 0–3 min before and 12–15 min after the start of the NBI27914 application. The frequency and amplitude of sIPSCs were analyzed using the Mini Analysis Program (Synaptosoft). Data are expressed as means ± standard error of the mean. Statistical analyses were performed using GraphPad Prism version 6. Differences with P < 0.05 were considered statistically significant. The detailed materials/methods and datasets used and/or analyzed in this study are provided in Additional files 1, 2, respectively.

Fig. 1
figure1

Enhanced sIPSC frequency in VTA-projecting dlBNST neurons in rats exposed to CMS. a Stressor protocol. b Stress exposure schedule. c Schematic diagram of the sites for retrobead injections and patch clamp recordings (REC) from the neurons retrogradely labelled with retrobeads. df Representative traces (d), frequency (e), and amplitude (f) of sIPSCs in VTA-projecting dlBNST type III neurons in the NS and CMS groups. g, h The effects of NBI27914 on the frequency of sIPSCs in VTA-projecting dlBNST type III neurons in the NS (g) and CMS groups (h). i, j The effects of NBI27914 on the amplitude of sIPSCs in VTA-projecting dlBNST type III neurons in the NS (i) and CMS groups (j). Data are expressed as means ± standard error of the mean. ***P < 0.001 (unpaired t-test)

Results

The frequency of sIPSCs in VTA-projecting dlBNST type III neurons was significantly higher in the CMS group (n = 41) than in the NS group (n = 39) (2.30 ± 0.28 (CMS) vs. 1.06 ± 0.22 (NS) Hz, t78 = 3.494, P = 0.0008, unpaired t-test; Fig. 1d, e). The sIPSC amplitude did not significantly differ between the CMS and NS groups (34.41 ± 2.32 (CMS) vs. 30.04 ± 2.88 (NS) pA t78 = 1.186, P = 0.2391, unpaired t-test; Fig. 1d, f). There are no differences of cellular properties of recorded neurons between the CMS and NS groups (Additional file 3).

After the recordings of the basal levels of sIPSCs, the effect of NBI27914 on sIPSCs in VTA-projecting dlBNST type III neurons was examined in some neurons (17/41 and 13/39 neurons in the CMS and NS groups, respectively). In the CMS group, bath-application of NBI27914 tended to decrease the frequency and amplitude of sIPSCs, although the effects were not significant (frequency: 3.27 ± 0.50 (pre) to 2.72 ± 0.56 (NBI) Hz, t16 = 1.415, P = 0.1763, paired t-test, Fig. 1h; amplitude: 37.12 ± 3.98 (pre) vs. 34.15 ± 3.75 (NBI) pA, t16 = 1.791, P = 0.0922, paired t-test, Fig. 1j). In the NS group, NBI27914 did not change the frequency and amplitude of sIPSCs (frequency: 1.67 ± 0.48 (pre) vs. 1.67 ± 0.61 (NBI) Hz, t12 = 0.007, P = 0.9949, paired t-test, Fig. 1g; amplitude: 41.18 ± 6.19 (pre) vs. 43.80 ± 9.32 (NBI) pA, t12 = 0.447, P = 0.6629, paired t-test, Fig. 1i).

Discussion

The present study revealed that the frequency of sIPSCs in VTA-projecting dlBNST neurons was enhanced in rats exposed to CMS, an animal model of depression, as observed in our previous study using an animal model of chronic pain [8]. Enhanced inhibitory transmission to VTA-projecting dlBNST neurons may be a neuronal mechanism common to depression and chronic pain that causes depression-like behaviors. Recently, Pati et al. reported that the frequency of sIPSCs in dlBNST neurons projecting to the VTA/lateral hypothalamus was enhanced in an animal model of alcohol withdrawal [11]. Enhanced inhibitory transmission to VTA-projecting dlBNST neurons may also be involved in alcohol withdrawal-induced depression.

We previously demonstrated that CRF selectively depolarizes dlBNST type II neurons [12] and increases the inhibitory synaptic inputs to dlBNST type III neurons [13]. Additionally, Marcinkiewcz et al. reported that CRF neurons in the BNST form local GABAergic synapses with BNST neurons that project to the VTA and mediate fear- and anxiety-like behaviors [14]. These findings suggest that at least one of the possible sources of inputs to VTA-projecting dlBNST neurons are dlBNST intrinsic neurons.

We previously reported that enhanced inhibitory synaptic inputs to VTA-projecting dlBNST neurons observed in the animal model of chronic pain were suppressed by NBI27914, a CRF type I receptor antagonist [8]. However, the current study showed that NBI27914 tended to suppress the CMS-induced facilitation of inhibitory synaptic inputs to VTA-projecting dlBNST neurons, but the effects were not significant, suggesting the different neuroplastic mechanisms for the enhancement of inhibitory synaptic inputs to VTA-projecting dlBNST neurons between CMS and chronic pain. Recently, Normandeau et al. reported that neurotensin and CRF co-acted to increase inhibitory synaptic transmission in the dlBNST of non-stressed rats and that CMS bolstered this potentiation through an enhanced contribution of neurotensin over CRF [15]. These findings suggest that neurotensin may be involved in the CMS-induced facilitation of inhibitory synaptic inputs to VTA-projecting dlBNST neurons. Further studies are needed to elucidate what kinds of neurotransmitters and/or neuropeptides are involved in the CMS-induced facilitation of inhibitory synaptic inputs to VTA-projecting dlBNST neurons.

Optogenetic activation of VTA-projecting BNST neuron terminals has been shown to produce rewarding effects in a real-time place preference test [16, 17]. Most VTA-projecting BNST neurons are GABAergic neurons that preferentially form synapses on VTA GABAergic neurons [9]. Thus, activation of VTA-projecting BNST neurons should promote VTA dopaminergic neuron activity by inhibiting the VTA GABAergic neurons that negatively regulate VTA dopaminergic neurons. In this context, enhanced inhibitory synaptic inputs to VTA-projecting BNST neurons may activate VTA GABAergic neurons via a disinhibition mechanism, thereby inhibiting VTA dopaminergic neurons. Suppression of the mesolimbic dopaminergic system may cause anhedonia in animal models of depression and chronic pain.

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its additional information files.

Abbreviations

BNST:

Bed nucleus of the stria terminalis

CMS:

Chronic mild stress

CRF:

Corticotropin-releasing factor

dlBNST:

Dorsolateral bed nucleus of the stria terminalis

NAc:

Nucleus accumbens

NS:

No stress

sIPSC:

Spontaneous inhibitory postsynaptic current

VTA:

Ventral tegmental area

References

  1. 1.

    Bair MJ, Robinson RL, Katon W, Kroenke K. Depression and pain comorbidity. Arch Intern Med. 2003;163:2433–45.

    Article  Google Scholar 

  2. 2.

    Yalcin I, Barthas F, Barrot M. Emotional consequences of neuropathic pain: insight from preclinical studies. Neurosci Biobehav Rev. 2014;47:154–64.

    Article  Google Scholar 

  3. 3.

    Nestler EJ, Carlezon WA Jr. The mesolimbic dopamine reward circuit in depression. Biol Psychiatry. 2006;59:1151–9.

    CAS  Article  Google Scholar 

  4. 4.

    Minami S, Satoyoshi H, Ide S, Inoue T, Yoshioka M, Minami M. Suppression of reward-induced dopamine release in the nucleus accumbens in animal models of depression: differential responses to drug treatment. Neurosci Lett. 2017;650:72–6.

    CAS  Article  Google Scholar 

  5. 5.

    Bessa JM, Morais M, Marques F, Pinto L, Palha JA, Almeida OF, Sousa N. Stress-induced anhedonia is associated with hypertrophy of medium spiny neurons of the nucleus accumbens. Transl Psychiatry. 2013;3:e266.

    CAS  Article  Google Scholar 

  6. 6.

    Kato T, Ide S, Minami M. Pain relief induces dopamine release in the rat nucleus accumbens during the early but not late phase of neuropathic pain. Neurosci Lett. 2016;629:73–8.

    CAS  Article  Google Scholar 

  7. 7.

    Asaoka Y, Kato T, Ide S, Amano T, Minami M. Pregabalin induces conditioned place preference in the rat during the early, but not late, stage of neuropathic pain. Neurosci Lett. 2018;668:133–7.

    CAS  Article  Google Scholar 

  8. 8.

    Takahashi D, Asaoka Y, Kimura K, Hara R, Arakaki S, Sakasai K, Suzuki H, Yamauchi N, Nomura H, Amano T, Minami M. Tonic suppression of the mesolimbic dopaminergic system by enhanced corticotropin-releasing factor signaling within the bed nucleus of the stria terminalis in chronic pain model rats. J Neurosci. 2019;39:8376–85.

    CAS  Article  Google Scholar 

  9. 9.

    Kudo T, Uchigashima M, Miyazaki T, Konno K, Yamasaki M, Yanagawa Y, Minami M, Watanabe M. Three types of neurochemical projection from the bed nucleus of the stria terminalis to the ventral tegmental area in adult mice. J Neurosci. 2012;32:18035–46.

    CAS  Article  Google Scholar 

  10. 10.

    Yamauchi N, Takahashi D, Sugimura YK, Kato F, Amano T, Minami M. Activation of the neural pathway from the dorsolateral bed nucleus of the stria terminalis to the central amygdala induces anxiety-like behaviors. Eur J Neurosci. 2018;48:3052–61.

    Article  Google Scholar 

  11. 11.

    Pati D, Marcinkiewcz CA, DiBerto JF, Cogan ES, McElligott ZA, Kash TL. Chronic intermittent ethanol exposure dysregulates a GABAergic microcircuit in the bed nucleus of the stria terminalis. Neuropharmacology. 2020;16:107759.

    Article  Google Scholar 

  12. 12.

    Ide S, Hara T, Ohno A, Tamano R, Koseki K, Naka T, Maruyama C, Kaneda K, Yoshioka M, Minami M. Opposing roles of corticotropin-releasing factor and neuropeptide Y within the dorsolateral bed nucleus of the stria terminalis in the negative affective component of pain in rats. J Neurosci. 2013;33:5881–94.

    CAS  Article  Google Scholar 

  13. 13.

    Nagano Y, Kaneda K, Maruyama C, Ide S, Kato F, Minami M. Corticotropin-releasing factor enhances inhibitory synaptic transmission to type III neurons in the bed nucleus of the stria terminalis. Neurosci Lett. 2015;600:56–61.

    CAS  Article  Google Scholar 

  14. 14.

    Marcinkiewcz CA, Mazzone CM, D’Agostino G, Halladay LR, Hardaway JA, DiBerto JF, Navarro M, Burnham N, Cristiano C, Dorrier CE, Tipton GJ, Ramakrishnan C, Kozicz T, Deisseroth K, Thiele TE, McElligott ZA, Holmes A, Heisler LK, Kash TL. Serotonin engages an anxiety and fear-promoting circuit in the extended amygdala. Nature. 2016;537:97–101.

    CAS  Article  Google Scholar 

  15. 15.

    Normandeau CP, Ventura-Silva AP, Hawken ER, Angelis S, Sjaarda C, Liu X, Pêgo JM, Dumont ÉC. A key role for neurotensin in chronic-stress-induced anxiety-like behavior in rats. Neuropsychopharmacology. 2018;43:285–93.

    CAS  Article  Google Scholar 

  16. 16.

    Kim SY, Adhikari A, Lee SY, Marshel JH, Kim CK, Mallory CS, Lo M, Pak S, Mattis J, Lim BK, Malenka RC, Warden MR, Neve R, Tye KM, Deisseroth K. Diverging neural pathways assemble a behavioural state from separable features in anxiety. Nature. 2013;496:219–23.

    CAS  Article  Google Scholar 

  17. 17.

    Jennings JH, Sparta DR, Stamatakis AM, Ung RL, Pleil KE, Kash TL, Stuber GD. Distinct extended amygdala circuits for divergent motivational states. Nature. 2013;496:224–8.

    CAS  Article  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This study was supported by Grant-in-Aid for Scientific Research (B) (M.M., 20H03389) and Challenging Research (Exploratory) (M.M., 19K22477) from the Japan Society for the Promotion of Science (JSPS). This research was also supported by Japan Agency for Medical Research and Development (AMED) under Grant number 20gm0910012 (M.M.).

Author information

Affiliations

Authors

Contributions

RH, TA and MM designed the experiments and prepared the manuscript. RH, DT and TT performed the experiments. RH and TA analyzed the data. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Masabumi Minami.

Ethics declarations

Ethics approval and consent to participate

All experiments were conducted with the approval of the Hokkaido University Institutional Animal Care and Use Committee. All efforts were made to minimize the number and suffering of animals used in the experiments.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher's Note

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

Supplementary information

Additional file 1.

Detailed materials/methods.

Additional file 2.

Datasets used and/or analyzed during the current study.

Additional file 3.

Cellular properties of recorded neurons in the NS and CMS groups.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hara, R., Takahashi, D., Takehara, T. et al. Inhibitory synaptic transmissions to the bed nucleus of the stria terminalis neurons projecting to the ventral tegmental area are enhanced in rats exposed to chronic mild stress. Mol Brain 13, 139 (2020). https://doi.org/10.1186/s13041-020-00684-4

Download citation

Keywords

  • Bed nucleus of the stria terminalis
  • Chronic mild stress
  • Chronic pain
  • Depression