Synaptic contacts of vesicular glutamate transporter 2 fibres on chemically identified neurons of the hypothalamic suprachiasmatic nucleus of the rat

Abstract

The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore, the purpose of the present study was to examine the relationship between the afferent glutamatergic axon terminals and the vasoactive intestinal polypeptide (VIP)-, arginine-vasopressin (AVP)- and gamma-aminobutyric acid (GABA)-positive neurons of the SCN. Glutamatergic elements were revealed via immunocytochemical double-labelling for vesicular glutamate transporter type 1 (VGluT1) and type 2 (VGluT2), and brain sections were imaged via confocal laser-scanning microscopy and electron microscopy. Numerous VGluT2-immunoreactive axons were observed to be in synaptic contact with VIP- and GABA-positive neurons, and only a few synapses were detected between VGluT2 boutons and AVP neurons. VGluT1 axon terminals exhibiting very moderate distribution in this cell group were observed to be in synaptic contact with chemically unidentified neurons. The findings provide the first morphological data on the termination of presumed glutamatergic fibres on chemically identified neurons of the rat SCN, and indicate that all three prominent cell types of the cell group receive glutamatergic afferents.

Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells

“…Both VGLUT2-and PACAP-positive nerve terminals have been shown to innervate VIP-positive cells in the ventrolateral retinorecipient area of the SCN (Kiss et al 2008;Sanggaard et al 2003). There is evidence that VIP plays a role in photic entrainment, coordinating daily rhythms in the SCN by synchronizing pace-making neurons; moreover, expression of VIP in the SCN is directly regulated by light (reviewed in Piggins and Cutler 2003).…”

“…Immunohistochemical localization of VGLUT2 was accomplished using a polyclonal anti-VGLUT2 antibody raised in guinea-pig (code no. : AB5907; Chemicon International, Temecula, CA, USA) (Hrabovszky et al 2005;Kiss et al 2008). A previously characterized mouse monoclonal anti-PACAP antibody (code no.…”

“…Previous studies in rodents have demonstrated the presence of VGLUT2 mRNA and protein in RGCs (Fujiyama et al 2003;Johnson et al 2004Johnson et al , 2007Stella et al 2008) and colocalization of VGLUT2 and melanopsin in ipRGCs of the mouse retina (Johnson et al 2007). VGLUT2 has been identified in visual target areas of the CNS (Fujiyama et al 2003;Kaneko et al 2002;Kiss et al 2007Kiss et al , 2008Land et al 2004;Sakata-Haga et al 2001;Varoqui et al 2002), but not shown in distinct target areas of the ipRGCs. PACAP, which in the retina is exclusively co-localized with melanopsin in ipRGCs, is found in retinal terminals in brain areas involved in NIF functions (Hannibal and Fahrenkrug 2004).…”

Phase Resetting of the Mammalian Circadian Clock Relies on a Rapid Shift of a Small Population of Pacemaker Neurons

“…The retinohypothalamic tract (RHT) is the major photic input pathway to the SCN [25] and projects predominantly to cells in the ventrolateral SCN [26]–[28] with glutamatergic and PACAP-containing fibers [25], [29]. The ventral (or core) cells of the SCN contain vasoactive intestinal peptide (VIP), gastrin-releasing peptide (GRP) and substance P (SP), which are colocalized to certain degrees [30]–[34].…”

KATP Channels Mediate Differential Metabolic Responses to Glucose Shortage of the Dorsomedial and Ventrolateral Oscillators in the Central Clock

“…For immunofluorescence staining, sections (20 µm) were washed for 20–30 min in PBS and then incubated overnight at 4 °C in PBS containing 2% serum, 0.3% Triton X-100, and primary antibodies against Kir6.2 (rabbit anti-Kir6.2; 1:500; APC-020; Alomone Labs, Jerusalem, Israel), AVP (guinea pig anti-AVP; 1:500; T-5048; Peninsula Laboratories, San Carlos, CA, USA) 48 , VIP (guinea pig anti-VIP; 1:500; T-5030; Peninsula Laboratories, San Carlos, CA, USA) 49 , GRP (goat anti-GRP; 1:500; sc-7788; Santa Cruz, CA, USA) 49 , neurophysin 2 (goat anti-neurophysin 2; 1:500; sc-27093; Santa Cruz, CA, USA) 50 , somatostatin (mouse anti-somatostatin; 1:500; sc-74556; Santa Cruz, CA, USA) 51 , vesicular glutamate transporter type 2 (vGluT2) (guinea pig anti-vGluT2; 1:500; AB2251; Millipore, Temecula, CA, USA) 52 , serotonin transporter (SERT) (mouse anti-SERT; 1:500, MAB1564; Millipore, Temecula, CA, USA) 53 , or NPY (guinea pig anti-NPY; 1:500, AB10341; Abcam, Cambridge, MA, USA) 54 . Sections were then treated with respective Alexa Fluor secondary antibodies 488 or 568 (diluted 1:300; Molecular Probes, Eugene, OR, USA) and Hoechst 33342 (B-2261; Sigma, St. Louis, MO, USA) for 1 hr at room temperature.…”