Arginine-Vasopressin Expressing Neurons in the Murine Suprachiasmatic Nucleus Exhibit a Circadian Rhythm in Network Coherence In Vivo

Abstract

The Suprachiasmatic Nucleus (SCN) is composed of functionally distinct sub-populations of GABAergic neurons such as vasoactive intestinal polypeptide (VIP)-, arginine vasopressin (AVP)-, gastrin releasing peptide (GRP)-, and neuromedin S (NMS)-expressing neurons which form a neural network responsible for synchronizing most physiological and behavioral circadian rhythms in mammals. To date, little is known regarding which aspects of SCN rhythmicity are generated by individual SCN neurons or neuronal sub-populations and which aspects result from neuronal interaction within a network. In this study, we address this question utilizing in vivo miniaturized microscopy to measure fluorescent GCaMP-mediated calcium dynamics in AVP neurons in the intact SCN of awake, behaving mice. This approach permits analysis of rhythms of single cells, populations, and correlational analysis among groups of AVP neurons in a field of view across the circadian and diurnal day and night. We report that AVP neurons in the murine SCN exhibit a periodic oscillatory increase in calcium of approximately 14 seconds across the day and night, in both constant darkness and under a 12:12 light-dark (LD) cycle. Using in vivo optogentically-targeted single unit activity recording, we demonstrated that these slow calcium waves are likely the result of burst-firing characteristic of AVP neurons previously reported for other brain regions. Rhythmicity analysis of several fluorescence measures suggests that individual AVP neurons exhibit unstable and stochastic rhythms, with approximately 30% of the neurons rhythmic during any given day across lighting conditions, and weak or absent rhythmicity at the population level. Network-level cross-correlational analysis revealed that coherence among neuron pairs also exhibited stochastic rhythms with about 25% of pairs rhythmic at any time. Notably, this analysis revealed a stronger rhythm at the population level than was observed in single cell analysis. The peak time of maximal coherence among AVP neuronal pairs occurs between CT/ZT 6 and 9, coinciding with the timing of maximal neuronal activity with the SCN as a whole. These results are the first to demonstrate robust circadian variation in the coordination between apparently weakly rhythmic or arrhythmic neurons suggesting that, for AVP neurons, interactions between neurons in the SCN are more influential than individual or single subpopulation activity in the regulation of mammalian circadian rhythms.

Nested calcium dynamics support daily cell unity and diversity in the suprachiasmatic nuclei of free-behaving mice

“…Since the SCN are composed of multiple neuronal subtypes, based on gene expression profiles or neuropeptide content (31)(32)(33)(34)(35), the question arises as to whether neurons exhibiting the same Ca 2+ i code belong to the same subtype and/or eventually constitute functional units gating specific body rhythms (13,(36)(37)(38)(39). In a parallel study, Stowie et al investigated circadian Ca 2+ i rhythmicity in arginine vasopressin-expressing SCN neurons in vivo (40). Further experiments implementing this cell typespecific approach will be necessary to monitor and manipulate Ca 2+ i signaling in identified SCN neurons coupled to specific circadian outputs.…”

Leptin receptor neurons in the dorsomedial hypothalamus input to the circadian feeding network

“…3B): (i) the "tonic calcium signal" that is the overall intensity of the fluorescence normalized to a 24-hour moving average and (ii) the "phasic calcium signal" that is the acute calcium signal increases above the baseline during each recording session as a proxy of dynamic neuron bursts, normalized to the average of dark phase (Fig. 3B) (66)(67)(68). We analyzed both of these readouts using two separate methods that yielded similar results (Fig.…”

A leptin-responsive hypothalamic circuit inputs to the circadian feeding network

“…The "tonic calcium signal" which is the overall intensity of the fluorescence normalized to 24-hour moving average and, 2. The "phasic calcium signal" which is the acute calcium signal increases above the baseline during each recording session as a proxy of dynamic neuron bursts, (Fig 3B, [62][63][64] . During SF, both types of calcium readouts from the DMH LepR neurons developed responses that predicted the meal time (Fig 3E-O).…”