Scientific Report 2005

Molecular Biology

Molecular Biology of Sleep

L. de Lecea, C. Suzuki, C. Pañeda, B. Boutrel,* R. Winsky-Sommerer, A. Coda, S. Huitrón-Reséndiz,* A.J. Roberts,* J.G. Sutcliffe, G.F. Koob,* S.J. Henriksen*

* Molecular and Integrative Neurosciences Department, Scripps Research

Our goal is to understand the cellular and molecular components that modulate cortical activity and sleep. In particular, we focus on the characterization of neuropeptides first described by our group: cortistatin and the hypocretins.

Cortistatin

Cortistatin is a neuropeptide expressed in the cerebral cortex. Of its 14 residues, 11 also occur in the neuropeptide somatostatin. However, cortistatin and somatostatin have different physiologic functions. Cortistatin is neuroinhibitory and promotes sleep.

We generated mice deficient in cortistatin and determined their behavioral profile in collaboration with A.J. Roberts, Molecular and Integrative Neurosciences Department. Because cortistatin has anticonvulsant activity, we tested seizure susceptibility in cortistatin-deficient mice. We also did gene array studies to determine the consequences of cortistatin deficiency in mice lacking the gene for this neuropeptide. Our results suggest that cortistatin has multiple functions in the maintenance of cortical excitability.

The Hypocretins

The hypocretins, 2 neuropeptides derived from the same precursor, are produced in a few thousand cells in the lateral part of the hypothalamus. The hypocretins are key molecules for the stability of the states of vigilance. Lack of hypocretin peptides or hypocretin-producing neurons produces narcolepsy, a sleep disorder characterized by uninvited intrusions of sleep into wakefulness. Patients with narcolepsy experience excessive daytime sleepiness and cataplexy, a sudden loss of muscle tone upon certain stimuli. Recent studies indicated that patients with narcolepsy lack hypocretin-expressing cells, suggesting that narcolepsy is a neurodegenerative disease of the hypocretinergic system.

In anatomic and electrophysiologic experiments, we found that neurons expressing hypocretin are contacted by neurons expressing corticotropin-releasing factor (CRF), a major component of the stress response. Hypocretin neurons contain CRF receptors. Intracellular recordings in hypothalamic slices from transgenic mice that express green fluorescent protein under the control of the hypocretin promoter indicated that CRF depolarizes hypocretin neurons through the CRF 1 receptor. Further, hypocretin neurons are not activated upon stress in mice that lack the gene for this receptor. These data suggest a close association between the CRF and hypocretin systems in the acute stress response.

Because CRF is involved in addiction and because hypocretin neurons project to key areas involved in brain reward, we hypothesized that hypocretin neurons might be involved in addiction-related behaviors. We found that hypocretin-1 leads to the reinstatement of previously extinguished cocaine-seeking behavior but does not alter cocaine intake in rats. In collaboration with P.J. Kenny and A. Markou, Molecular and Integrative Neurosciences Department, we discovered that hypocretin-1 negatively regulates the activity of brain reward circuitries. Hypocretin-induced reinstatement of cocaine seeking can be prevented by simultaneous blockade of noradrenergic and CRF systems but not by blockade of either system alone. These findings reveal a previously unidentified role for hypocretins in drug craving and relapse behavior. Moreover, hypocretins may drive drug seeking through induction of a negative affective state by activation of stress pathways in the brain.

Neuropeptide S

Neuropeptide S is a newly discovered neuropeptide expressed prominently in a few hundred neurons in the area near the locus coeruleus. We found that infusion of neuropeptide S into the brain ventricles in mice dramatically enhanced wakefulness and suppressed anxiety. The neuropeptide activated several brain nuclei related to arousal. We showed that neurons expressing neuropeptide S project to and depolarize neurons expressing hypocretin. Our data strongly suggest that neuropeptide S is an important modulator of sleep and waking.

Publications

de Lecea, L. Reverse genetics and the study of sleep. In: Sleep: Circuits and Functions. Luppi, P.-H. (Ed.). CRC Press, Boca Raton, FL, 2004, p. 109.

de Lecea, L., Sutcliffe, J.G. The hypocretins and sleep. FEBS J., in press.

de Lecea, L., Sutcliffe, J.G. (Eds.) Hypocretins: Integrators of Physiological Functions. Springer, New York, 2005.

Huitrón-Reséndiz, S., Kristensen, M.P., Sánchez-Alavez, M., Clark, S.D., Grupke, S.L., Tyler, C., Suzuki, C., Nothacker, H.P., Civelli, O., Criado, J.R., Henriksen, S.J., Leonard, C.S., de Lecea, L. Urotensin II modulates rapid eye movement sleep through activation of brainstem cholinergic neurons. J. Neurosci. 25:5465, 2005.

Levine, A.S., Winsky-Sommerer, R., Huitrón-Reséndiz, S., Grace, M.K., de Lecea, L. Injection of neuropeptide W into paraventricular nucleus of hypothalamus increases food intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 288:R1727, 2005.

Martin, G., Guadaño-Ferraz, A., Morte, B., Ahmed, S., Koob, G.F., de Lecea, L. Siggins, G.R. Chronic morphine treatment alters N-methyl-D-aspartate receptors in freshly isolated neurons from nucleus accumbens. J. Pharmacol. Exp. Ther. 311:265-73, 2004.

Pañeda, C., Winsky-Sommerer, R., Boutrel, B., de Lecea, L. The corticotropin-releasing factor-hypocretin connection: implications in stress response and addiction. Drug News Perspect. 18:250, 2005.

Spier, A.D., Fabre, V., de Lecea, L. Cortistatin radioligand binding in wild-type and somatostatin receptor-deficient mouse brain. Regul. Pept. 124:179, 2005.

Sutcliffe, J.G., de Lecea L. Not asleep, not quite awake. Nat. Med. 10:673, 2004.

Tallent, M.K., Fabre, V., Qiu, C., Calbet, M., Lamp, T., Baratta, M.V., Suzuki, C., Siggins, G.R., Henriksen, S.J., Criado, J.R., Roberts, A., de Lecea, L., Cortistatin overexpression in transgenic mice produces deficits in synaptic plasticity and learning. Mol. Cell. Neurosci., in press.

Ureña, J.M., La Torre, A., Martìnez, A., Lowenstein, E., Franco, N., Winsky-Sommerer, R., Fontana, X., Casaroli-Marano, R., Ibáñez-Sabio, M.A., Pascual, M., del Rio, J.A., de Lecea, L., Soriano, E. Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain. J. Comp. Neurol. 490:119, 2005.

Winsky-Sommerer, R., Boutrel, B., de Lecea , L. Stress and arousal: the corticotropin-releasing factor/hypocretin circuitry. J. Mol. Neurobiol., in press.

Winsky-Sommerer, R., Yamanaka, A., Diano, S., Borok, E., Roberts, A., Sakurai, T., Kilduff, T.S., Horvath, T.L., de Lecea, L. Interaction between the corticotropin-releasing factor system and hypocretins (orexins): a novel circuit mediating stress response. J. Neurosci. 24:11439, 2004.

Xu, Y., Reinscheid, R.R., Huitrón-Reséndiz, S., Clark, S.D., Wang, Z., Lin, S.H., Brucher, F.A., Zeng, J., Ly, H.K., Henriksen, S.J., de Lecea, L., Civelli, O. Neuropeptide S: a novel neuropeptide promoting arousal and anxiolytic-like effects. Neuron 43:487, 2004.