In the right place and at the right time : cellular and functional neuroanatomy of endocannabinoids and neuropeptides

A plethora of chemical signals, including endocannabinoids and neuropeptides, enable neuronal communication beyond the classical neurotransmitters. Many of these molecules are also expressed during (early) brain development, helping neurons to mature and neuronal circuits to take form. This thesis focuses on the roles of these “unusual transmitters” in (the establishment of) brain circuits. Besides neurons, the brain also consists of glial cells: astrocytes, microglia, and oligodendrocytes. Glial cells play invaluable roles in not only the adult brain, but also in the developing nervous system. Endocannabinoid signaling promotes several processes in neuronal development, including axonal growth and elongation mediated by the growth cone. In study I, we inhibited the degrading enzyme of the endocannabinoid 2-arachidonoylglycerol (2-AG), which resulted in errors in axonal fasciculation and premature maturation of oligodendrocytes within axonal bundles. Neuronal growth cones were repulsed via interactions of the axon guidance cues Slit and Robo. Thus, cannabinoid type 1 receptor (CB1R) signaling repositioned Robo1 to neuronal growth cones on the one hand, while CB2R signaling induced excess production of Slit2 in oligodendrocytes on the other. Taken together, this indicates that endocannabinoids regulate downstream Slit/Robo signaling in embryonic development.

In the first two postnatal weeks, the newly established brain circuits undergo extensive remodeling. In study II, we discovered that the neuropeptide galanin is transiently expressed in cells in the somatosensory thalamus during this time, which was confirmed in the galanin-Cre::tdTomato mouse. We designed a 3D-printed extension for the stereotactic apparatus to perform viral circuit tracing in 7-day old pups, at a time of active Cre expression. We established that the cells that transiently express galanin are neurons in the ventrobasal nucleus of the thalamus (VB) projecting towards the somatosensory cortex. The robust expression of galanin in VB soma and the discovery of galanin receptors in the VB hints at potential somatodendritic release of galanin, which could be involved in the postnatal refinement of the VB.

A brain region known for its diverse expression of neuropeptides, including galanin, is the hypothalamus. In study III, we attempted to analyze the molecular diversity of the hypothalamus using single-cell sequencing. This resulted in 62 neuronal clusters segregated by enzymes involved in the turnover of neurotransmitters and by neuropeptides. Using amongst others viral circuit tracing, we investigated how hypothalamic neurons functionally integrate in hitherto unknown brain circuits. In study III, we could place a novel dopaminergic neuronal subtype in a brain circuit receiving neuropeptidergic inputs from the suprachiasmatic nucleus and projecting towards the median eminence. These neurons are probably involved in the circadian control of release of dopamine which controls the secretion of prolactin. In study IV, we uncovered a new neuronal stress circuitry increasing cortical alertness. It links stress-responsive hypothalamic neurons via volume transmission of the neurotrophin ciliary neurotrophic factor (CNTF) in the cerebrospinal fluid to the noradrenergic neurons in the pontine locus coeruleus. These neurons in turn project to the prefrontal cortex inducing long-lasting excitability in response to acute stress.

List of scientific papers

I. Alán Alpár, Giuseppe Tortoriello, Daniela Calvigioni, Micah J. Niphakis, Ivan Milenkovic, Joanne Bakker, Gary A. Cameron, János Hanics, Claudia V. Morris, János Fuzik, Gabor G. Kovacs, Benjamin F. Cravatt, John G. Parnavelas, William D. Andrews, Yasmin L. Hurd, Erik Keimpema, and Tibor Harkany. Endocannabinoids modulate cortical development by configuring Slit2/Robo1 signalling. Nature Communications. 2014, 5, 4421.
https://doi.org/10.1038/ncomms5421

II. Joanne Bakker, Simon Steffens, Swapnali S. Barde, Csaba Adori, Sabah Rehman, Susanne Brunner, Barbara Kofler, Konstantinos Meletis, Tibor Harkany and Tomas G.M. Hökfelt. Galanin is transiently expressed in the neonatal somatosensory thalamus. [Manuscript]

III. Roman A. Romanov, Amit Zeisel, Joanne Bakker, Fatima Girach, Arash Hellysaz, Raju Tomer, Alán Alpár, Jan Mulder, Frédéric Clotman, Erik Keimpema, Brian Hsueh, Ailey K. Crow, Henrik Martens, Christian Schwindling, Daniela Calvigioni, Jaideep S. Bains, Zoltán Máté, Gábor Szabó, Yuchio Yanagawa, Ming-Dong Zhang, Andre Rendeiro, Matthias Farlik, Mathias Uhlén, Peer Wulff, Christoph Bock, Christian Broberger, Karl Deisseroth, Tomas Hökfelt, Sten Linnarsson, Tamas L. Horvath & Tibor Harkany. Molecular interrogation of hypothalamic organization reveals distinct dopamine neuronal subtypes. Nature Neuroscience. 2017, 20, 176-188.
https://doi.org/10.1038/nn.4462

IV. Alán Alpár, Péter Zahola, János Hanics, Zsófia Hevesi, Solomiia Korchynska, Marco Benevento, Christian Pifl, Gergely Zachar, Jessica Perugini, Ilenia Severi, Patrick Leitgeb, Joanne Bakker, Andras G. Miklosi, Evgenii Tretiakov, Erik Keimpema, Gloria Arque, Ramon O. Tasan, Günther Sperk, Katarzyna Malenczyk, Zoltán Máté, Ferenc Erdélyi, Gábor Szabó, Gert Lubec, Miklós Palkovits, Antonio Giordano, Tomas G.M. Hökfelt, Roman A. Romanov, Tamas L. Horvath & Tibor Harkany. Hypothalamic CNTF volume transmission shapes cortical noradrenergic excitability upon acute stress. The EMBO Journal. 2018, 37 (21).
https://doi.org/10.15252/embj.2018100087