Immunohistochemical evidence of serotoninergic regulation of vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus

Abstract

By applying a double-immunolabeling technique to preembedded tissue preparations, we demonstrated the existence of serotoninergic innervation to neurons containing vasoactive intestinal polypeptide (VIP) in the rat suprachiasmatic nucleus (SCN). Immunoreactivity for serotonin and VIP was revealed by the presence of diaminobenzidine (DAB) reaction products and silver-intensified DAB reaction products, respectively; in a further stage, the silver grains were substituted with gold particles. DAB reaction products were precipitated on the surface of vesicular structures, while gold particles were scattered diffusely throughout the neuroplasma at various densities. Serotoninergic axons were numerous and closely packed together, occasionally forming synaptic junctions with gold-labeled VIP-containing neurons. At these synaptic junctions, small vesicular structures accumulated to form a coat under the presynaptic membrane, and the postsynaptic membrane was lined with a homogeneous accumulation of fine deposits. This postsynaptic apparatus varied in appearance; some parts were flat and thin, while others were of irregular thickness. Serotoninergic fibers also formed synaptic junctions with unidentified neurons, in which postsynaptic membrane specialization was also observable. As VIP-containing neurons are known to be synapsed by somatostatin (SRIH)-containing neurons, their regulation must involve both serotonin and SRIH at least.

Ultrastructural Evidence for Neuronal Regulation of Growth Hormone Secretion

“…Van den Pol [50] used metallic intensification of the immunoreaction products to increase immunoreaction signals. Silver-gold intensifica tion of DAB chromogen was first reported by a Hungarian group [29], and was used by Liposits et al [28,30], Hisano et al [17] and Maegawa et al [31] for electron-microscopic distinction between two substantially different types of neurons in preembedding immunolabeling procedures: one type of neuron was detected by accumulation of metallic particles, and the other with DAB chromogen. We used this method in the present study.…”

“…This was fol lowed by a silver-gold intensification step for the DAB reactive products from the first stain. Then, the reaction for the second antigen was completed by applying ABC fol lowed by unintensified DAB [17,31]. Our effort was fo cused on preserving the antigenicity of the second antigen during the treatment for the first antigen.…”

Constructing the suprachiasmatic nucleus: a watchmaker's perspective on the central clockworks

“…ipRGC innervation is particularly prominent for Vip neurons in the vlSCN, reflecting their key role in light entrainment, though all SCN subdomains receive at least some direct ipRGC input in mammals (Ibata et al, 1989 ; Tanaka et al, 1993 ; Muscat et al, 2003 ; Hattar et al, 2006 ; Morin et al, 2006 ; Juarez et al, 2013 ). Other major afferents, such as IGL-derived Npy and 5HT from the raphe, also preferentially innervate the vlSCN in a closely overlapping pattern, with synaptic terminals seen on Vip neurons (Kiss et al, 1984 ; Bosler and Beaudet, 1985 ; Hisano et al, 1987 ; Abrahamson and Moore, 2001 ). Conversely, septal neurons project selectively to SCN Avp neurons (Oldfield et al, 1985 ).…”

Daily and Seasonal Rhythms

“…In addition to receiving inputs from retinal afferents of the RHT and GHT, VIP-containing neurons receive synaptic inputs from GABAergic neurons, somatostatin-containing neurons, and serotonergic neurons projecting from the midbrain raphe nuclei (326, 438).…”