Whole-Brain Monosynaptic Afferent Projections to the Cholecystokinin Neurons of the Suprachiasmatic Nucleus

Abstract

The suprachiasmatic nucleus (SCN) is the principal pacemaker driving the circadian rhythms of physiological behaviors. The SCN consists of distinct neurons expressing neuropeptides, including arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP), gastrin-releasing peptide (GRP), cholecystokinin (CCK), and so on. AVP, VIP, and GRP neurons receive light stimulation from the retina to synchronize endogenous circadian clocks with the solar day, whereas CCK neurons are not directly innervated by retinal ganglion cells and may be involved in the non-photic regulation of the circadian clock. To better understand the function of CCK neurons in non-photic circadian rhythm, it is vital to clarify the direct afferent inputs to CCK neurons in the SCN. Here, we utilized a recently developed rabies virus- and Cre/loxP-based, cell type-specific, retrograde tracing system to map and quantitatively analyze the whole-brain monosynaptic inputs to SCN CCK neurons. We found that SCN CCK neurons received direct inputs from 29 brain nuclei. Among these nuclei, paraventricular nucleus of the hypothalamus (PVH), paraventricular nucleus of the thalamus (PVT), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus, and seven other nuclei sent numerous inputs to CCK neurons. Moderate inputs originated from the zona incerta, periventricular hypothalamic nucleus, and five other nuclei. A few inputs to CCK neurons originated from the orbital frontal cortex, prelimbic cortex, cingulate cortex, claustrum, and seven other nuclei. In addition, SCN CCK neurons were preferentially innervated by AVP neurons of the ipsilateral PVH and SON rather than their contralateral counterpart, whereas the contralateral PVT sent more projections to CCK neurons than to its ipsilateral counterpart. Taken together, these results expand our knowledge of the specific innervation to mouse SCN CCK neurons and provide an important indication for further investigations on the function of CCK neurons.

Suprachiasmatic VIP neurons are required for normal circadian rhythmicity and comprised of molecularly distinct subpopulations

“…To this end, we also noted substantial input from several preoptic cell groups, suggesting a potential circuit substrate by which temperature sensitive neurons might modulate, and possibly entrain, SCN VIP neurons 56 . It is also useful to contrast our findings with those from a recent rabies-based tracing study 57 which showed that CCK-expressing SCN neurons, which are generally intermingled with, but are also distinct from, SCN VIP neurons, receive input from a large number of hypothalamic and preoptic structures, but not from the retina, and minimally from the IGL.…”

Whole-Brain Monosynaptic Inputs to Hypoglossal Motor Neurons in Mice

“…AAV2/9-CAG-DIO-TVA-GFP (1.7 9 10 13 genome copies/mL), AAV2/9-CAG-DIO-RG (6.8 9 10 12 genome copies/mL), and an EnvA-pseudotyped glycoprotein (RG)deleted and DsRed-expressing rabies virus (RV-EnvA-DG-DsRed, 5.0 9 10 8 fluorescence-forming units/mL) were purchased from BrainVTA (Wuhan, China) [21,22]. The two Cre-dependent AAVs were mixed at a 1:1 ratio in 100 nL as helper viruses for retrograde monosynaptic-tracing experiments.…”

“…Surgical procedures were performed as described in previous studies [22,23]. Briefly, mice were anesthetized with chloral hydrate (350 mg/kg, intraperitoneal) and placed in a stereotaxic apparatus.…”

“…To confirm that the neurons initially infected were cholinergic neurons in the 12N, we immunostained sections containing the 12N with primary antibodies to ChAT. To characterize the inputs and initially-infected neurons of the CeA and PSTN, we immunostained sections containing these areas with primary antibodies against c-aminobutyric acid (GABA) for the CeA and CR for the PSTN [21,22]. The sections were incubated overnight at 4°C in PBST (PBS with 0.3% Triton X-100 [v/v]) and with the following primary antibodies: goat anti-ChAT (1:500, cat.…”

Neurogenetic basis for circadian regulation of metabolism by the hypothalamus

“…Input to the SCN also arises from hypothalamic and brain stem areas, including the PVH and the preoptic area (Pickard 1982;Yuan et al 2018), although the functional relevance of most such bidirectional circuits remains largely unknown. Serotonergic input from the brain stem raphe nuclei is particularly dense to the SCN (Meyer-Bernstein and Morin 1996; Pickard and Rea 1997) and may be involved in modulating functions such as how light and locomotor activity rhythms convey entrainment signals to the SCN Marchant et al 1997).…”