Anatomical studies of the dopamine system in the hypothalamus and the pituitary gland

The hypothalamus is a small, evolutionarily conserved brain region, necessary for our survival as individuals and as a species. It collects various sensory inputs, process them to maintain homeostasis and to overcome stressors, and generates outputs that affect the autonomic nervous system, the endocrine system and somatomotor behaviors. Energy metabolism, fluid balance, thermoregulation, sleep, aggression and reproduction are examples of functions under direct and indirect hypothalamic control. The neurochemical basis for these regulations involve different neurotransmitters and neuromodulators. The catecholamine dopamine is highly associated with various hypothalamic functions and behaviors, and has early on been shown to be present in intrinsic hypothalamic populations as well as incoming axon terminals. It acts on two types of receptors, excitatory D1-type and inhibitory D2-type, of which both have been reported to be expressed in the hypothalamus.

To increase our understanding of these circuitries, this thesis aims to investigate the dopamine system in the hypothalamus, and the structures closely related to its inputs and outputs, namely the circumventricular organs and the pituitary. Immunohistochemical methods were used to generate a comprehensive distribution map of dopamine’s two main receptors, D1 and D2, and the neurochemical identity of these dopamine-receptor expressing cells were characterized. While the D2 receptor was widely expressed, D1 expression was found to be sparse. The suprachiasmatic nucleus, however, showed the contrary expression pattern. The D2 receptor could be localized to parvocellular neurons as well as endocrine cells of the pituitary. Little evidence for dopamine receptor expression on the magnocellular neurons could, however, be observed. Evidence for D1 receptor expression was also found in the subcommissural organ and a sub-cluster of ependymal cells in the third ventricle. Tuberoinfundibular dopamine (TIDA) neurons, which release dopamine in the portal vessels and thereby inhibit lactotrophs and prolactin release, were investigated in greater detail with regards to modulatory input, and morphological features. Anatomical substrate for innervation by serotonin and hypocretin/orexin on TIDA cell body and dendrites was identified together with electrophysiological evidence for excitation and suppression by hypocretin/orexin and serotonin or selective serotonin reuptake inhibitors, respectively.

Morphological studies of male mice and rat TIDA neurons were done on tissue section and marker filled neurons by means of immunohistochemistry. TIDA neurons were found to preferentially extend dendrites towards the third ventricle, possibly even into the ventricle. Axon terminals were found in the median eminence, but collateral branches oriented laterally could also be detected. An intermingling subcellular distribution of inhibitory and excitatory synapses, on somatic and dendritic level, was also identified. No significant differences could be observed in most morphological properties of mouse and rat TIDA neurons. However, rats exhibited a higher total number of TIDA neurons and a lower spine density than mice. Finally, the expression of three different calcium binding proteins, i.e. calbindin-D28k, calretinin and parvalbumin, were investigated within the arcuate nucleus. While both calbindin-D28k and calretinin could be detected in the arcuate nucleus, little evidence for parvalbumin expression was observed. None of the proteins were expressed in TIDA neurons or other investigated populations, except for proopiomelanocortin neurons that expressed both calbindin-D28k and calretinin. These neurons showed a rostrocaudal segregation of the two calcium binding proteins that resulted in two separate subpopulations.

Overall, the studies presented in this thesis reveal a previously unappreciated abundance of dopaminergic involvement in the hypothalamic circuitries which will increase our understanding of mammalian homeostatic and endocrine control.

List of scientific papers

I. Kylie S. Foo, Arash Hellysaz and Christian Broberger. (2014). Expression and colocalization patterns of calbindin-D28k, calretinin and parvalbumin in the rat hypothalamic arcuate nucleus. Journal of Chemical Neuroanatomy. 61:20–32.
https://doi.org/10.1016/j.jchemneu.2014.06.008

II. Arash Hellysaz, Marta Garo, Rachida Ammari, David J. Lyons and Christian Broberger. (2018). Morphological characterization of tuberoinfundibular dopamine (TIDA) neurons in rodents. [Manuscript]

III. David J. Lyons, Rachida Ammari, Arash Hellysaz and Christian Broberger. (2016). Serotonin and antidepressant SSRIs inhibit rat neuroendocrine dopamine neurons: Parallel actions in the lactotrophic axis. Journal of Neuroscience. 36:7392–7406.
https://doi.org/10.1523/JNEUROSCI.4061-15.2016

IV. David J. Lyons, Arash Hellysaz, Rachida Ammari and Christian Broberger. (2017). Hypocretin/orexin peptides excite rat neuroendocrine dopamine neurons through orexin 2 receptor-mediated activation of a mixed cation current. Scientific Reports. 7:41535.
https://doi.org/10.1038/srep41535

V. Arash Hellysaz, Alessandra Bonito-Oliva and Christian Broberger. (2018). Distribution and neurochemical characterization of dopamine D2 receptor expressing neurons in the hypothalamus and pituitary gland of Drd2-EGFP transgenic mice. [Manuscript]

VI. Arash Hellysaz, Qingyun Wen and Christian Broberger. (2018). Distribution and neurochemical characterization of dopamine D1 receptor expressing cells in the hypothalamus, circumventricular organs and pituitary gland in mice. [Manuscript]