Brain Structure and Function

, Volume 222, Issue 1, pp 515–537 | Cite as

Nucleus incertus promotes cortical desynchronization and behavioral arousal

  • Sherie MaEmail author
  • Giancarlo Allocca
  • Emma K. E. Ong-Pålsson
  • Caitlin E. Singleton
  • David Hawkes
  • Stuart J. McDougall
  • Spencer J. Williams
  • Ross A. D. Bathgate
  • Andrew L. GundlachEmail author
Original Article


Arousal and vigilance are essential for survival and relevant regulatory neural circuits lie within the brainstem, hypothalamus and forebrain. The nucleus incertus (NI) is a distinct site within the pontine periventricular gray, containing a substantial population of GABAergic neurons with long-range, ascending projections. Existing neuroanatomical data and functional studies in anesthetized rats, suggest the NI is a central component of a midline behavioral control network well positioned to modulate arousal, vigilance and exploratory navigation, yet none of these roles have been established experimentally. We used a chemogenetic approach—clozapine-N-oxide (CNO) activation of virally delivered excitatory hM3Dq-DREADDs—to activate the NI in rats and examined the behavioral and physiological effects, relative to effects in naïve rats and appropriate viral-treated controls. hM3Dq activation by CNO resulted in long-lasting depolarization of NI neurons with action potentials, in vitro. Peripheral injection of CNO significantly increased c-Fos immunoreactivity in the NI and promoted cortical electroencephalograph (EEG) desynchronization. These brain changes were associated with heightened arousal, and increased locomotor activity in the homecage and in a novel environment. Furthermore, NI activation altered responses in a fear conditioning paradigm, reflected by increased head-scanning, vigilant behaviors during conditioned fear recall. These findings provide direct evidence that the NI promotes general arousal via a broad behavioral activation circuit and support early hypotheses, based on its connectivity, that the NI is a modulator of cognition and attention, and emotional and motivated behaviors.


Arousal Nucleus incertus Cortical desynchronization Vigilance Locomotion 



The authors would like to thank Prof Bryan Roth (University of North Carolina, NC, USA) and Dr. Melanie White (ARMI, Monash University, VIC, Australia) for access to the DREADD construct and assistance in establishing the approach in our laboratory, respectively; and Prof Neil McNaughton (University of Otago, Dunedin, NZ) for valuable comments on the manuscript. This research was supported by project grants from the National Health and Medical Research Council (NHMRC) of Australia (1005988 and 1067522, A. L. G. and R. A. D. B.); a Grant from The Florey Institute of Neuroscience and Mental Health Foundation (A. L. G. and S. M.); a Grant from the Besen Family Foundation (A. L. G.); and by the Victorian Government Operational Infrastructure Support Program. S. J. W. is an Australian Research Council (ARC) Future Fellow. R. A. D. B. and A. L. G. are NHMRC (Australia) Senior Research Fellows. G.A. is the recipient of a Commonwealth of Australia International Postgraduate Research Scholarship (IPRS); and E. K. E. O-P. is the recipient of a University of Melbourne International Research Scholarship (MIFRS/MRS).

Supplementary material

429_2016_1230_MOESM1_ESM.mp4 (3.2 mb)
Online resource 1. NI activation is associated with persistent locomotor activity. Concurrent video recording of 2 × NI-hM3Dq rats (upper chambers) and 2 × NI-mCherry rats (lower chambers) in automated locomotor cells during a 3 h trial immediately after injection of CNO (3 mg/kg, i.p.). Video speed is 2 × normal. Flashes of light are infrared beams activated automatically by the locomotor cell software that are not detected by the rats. Time-lapsed video recordings of activity illustrate the persistent ambulation and ‘interrupted’ attempts to rest of NI-hM3Dq rats. (MP4 3294 kb)
429_2016_1230_MOESM2_ESM.mp4 (2.2 mb)
Online resource 2. NI activation is associated with increased risk-assessment and head-scanning behavior in response to learned potential threat. Concurrent video recording of 2 × NI-hM3Dq rats (lower chambers) and 2 × NI-mCherry rats (upper chambers) in operant chambers 24 h after fear conditioning where rats were trained to associate an audible tone (30 s, 80 dB, 5 kHz sine wave) with footshock (1 s, 0.7 mA). Video speed is 2 × normal. The video starts during the intertrial interval between tones 2-3, during which scanning behavior is exhibited by NI-hM3Dq rats, but infrequently by NI-mCherry rats that instead exhibit predominant freezing and immobility. A 30 s tone presentation (tone 3) is indicated by the presence of a small red LED on top of the chamber (not within view of the rats). During and after tone presentation, NI-hM3Dq rats exhibit high levels of head-scanning and risk assessment behavior. (MP4 2264 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Sherie Ma
    • 1
    • 2
    Email author
  • Giancarlo Allocca
    • 1
    • 2
  • Emma K. E. Ong-Pålsson
    • 1
    • 2
  • Caitlin E. Singleton
    • 1
  • David Hawkes
    • 1
    • 3
  • Stuart J. McDougall
    • 1
    • 2
  • Spencer J. Williams
    • 4
  • Ross A. D. Bathgate
    • 1
    • 2
    • 5
  • Andrew L. Gundlach
    • 1
    • 2
    • 6
    Email author
  1. 1.The Florey Institute of Neuroscience and Mental HealthParkvilleAustralia
  2. 2.Florey Department of Neuroscience and Mental HealthThe University of MelbourneParkvilleAustralia
  3. 3.Department of Pharmacology and TherapeuticsThe University of MelbourneParkvilleAustralia
  4. 4.School of Chemistry and Bio21 Molecular Science and Biotechnology InstituteThe University of MelbourneParkvilleAustralia
  5. 5.Department of Biochemistry and Molecular BiologyThe University of MelbourneParkvilleAustralia
  6. 6.Department of Anatomy and NeuroscienceThe University of MelbourneParkvilleAustralia

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