Arginine Vasopressin-Containing Neurons of the Suprachiasmatic Nucleus Project to CSF.

While it is well established that there are robust circadian rhythms of arginine vasopressin (AVP) in the cerebrospinal fluid (CSF), the route whereby the peptide reaches the CSF is not clear. A , AVP neurons constitute the largest fraction of the SCN neuronal population. Here, we show that processes of AVP-expressing SCN neurons cross the epithelium of the 3rd ventricular wall to reach the CSF (black arrows). Additionally, we report rostro-caudal differences in AVP neuron size and demonstrate that the localization of cells expressing the clock protein PER2 extend beyond the AVP population, thereby indicating that the size of this nucleus is somewhat larger than previously understood. B , Following lateral ventricle (LV) injection of cholera toxin ß subunit (CTß ; magenta) the retrograde tracer is seen in AVP neurons of the SCN, supporting the anatomical evidence that AVP neuronal processes directly contact the CSF.Arginine vasopressin (AVP) expressing neurons form the major population in the brain's circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN). They participate in inter-neuronal coupling and provide an output signal for synchronizing daily rhythms. AVP is present at high concentrations in the cerebrospinal fluid (CSF) and fluctuates on a circadian timescale. While it is assumed that rhythms in CSF AVP are of SCN origin, a route of communication between these compartments has not been delineated. Using immunochemistry (ICC) and cell filling techniques, we determine the morphology and location of AVP neurons in mouse and delineate their axonal and dendritic processes. Cholera toxin ß subunit (CTß) tracer injected into the lateral ventricle tests whether AVP neurons communicate with CSF. Most importantly, the results indicate that AVP neurons lie in close proximity to the third ventricle, and their processes cross the ventricular wall into the CSF. We also report that contrary to widely held assumptions, AVP neurons do not fully delineate the SCN borders as PER2 expression extends beyond the AVP region. Also, AVP neurons form a rostral prong originating in the SCN medial-most and ventral-most aspect. AVP is lacking in the mid-dorsal shell but does occur at the base of the SCN just above the optic tract. Finally, neurons of the rostral SCN are smaller than those lying caudally. These findings extend our understanding of AVP signaling potential, demonstrate the heterogeneity of AVP neurons, and highlight limits in using this peptide to delineate the mouse SCN.

Musashi-2 and related stem cell proteins in the mouse suprachiasmatic nucleus and their potential role in circadian rhythms.

BACKGROUND: The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus contains the master circadian clock of the body and an unusually large number of cells expressing stem cell-related proteins. These seemingly undifferentiated cells may serve in entrainment of the SCN circadian clock to light cycles or allow it to undergo neural plasticity important for modifying its rhythmic output signals. These cells may also proliferate and differentiate into neurons or glia in response to episodic stimuli or developmental events requiring alterations in the SCN's control of physiology and behavior. PROBLEM: To characterize expression of stem cell related proteins in the SCN and the effects of stem-like cells on circadian rhythms. METHODS: Explant cultures of mouse SCN were maintained in medium designed to promote survival and growth of stem cells but not neuronal cells. Several stem cell marker proteins including SRY-box containing gene 2 (SOX2), nestin, vimentin, octamer-binding protein 4 (OCT4), and Musashi RNA-binding protein 2 (MSI2) were identified by immunocytochemistry in histological sections from adult mouse SCN and in cultures of microdissected SCN. A bioinformatics analysis located potential SCN targets of MSI2 and related RNA-binding proteins. RESULTS: Cells expressing stem cell markers proliferated in culture. Immunostained brain sections and bioinformatics supported the view that MSI2 regulates immature properties of SCN neurons, potentially providing flexibility in SCN neural circuits. Explant cultures had ongoing mitotic activity, indicated by proliferating-cell nuclear antigen, and extensive cell loss shown by propidium iodide staining. Cells positive for vasoactive intestinal polypeptide (VIP) that are highly enriched in the SCN were diminished in explant cultures. Despite neuronal cell loss, tissue remained viable for over 7 weeks in culture, as shown by bioluminescence imaging of explants prepared from SCN of Per1::luc transgenic mice. The circadian rhythm in SCN gene expression persisted in brain slice cultures in stem cell medium. Prominent, widespread expression of RNA-binding protein MSI2 supported the importance of posttranscriptional regulation in SCN functions and provided further evidence of stem-like cells. CONCLUSION: The results show that the SCN retains properties of immature neurons and these properties persist in culture conditions suitable for stem cells, where the SCN stem-like cells also proliferate. These properties may allow adaptive circadian rhythm adjustments. Further exploration should examine stem-like cells of the SCN in vivo, how they may affect circadian rhythms, and whether MSI2 serves as a master regulator of SCN stem-like properties.