Glutamate‐like Immunoreactivity in Retinal Terminals of the Mouse Suprachiasmatic Nucleus

Abstract

With a view to identifying the neurotransmitter content of retinal terminals within the mouse suprachiasmatic nucleus, a highly specific antiserum to glutaraldehyde-coupled glutamate was used in a postembedding immunogold procedure at the ultrastructural level. Retinal terminals were identified by cholera toxin--horseradish peroxidase transported anterogradely from the retina and reacted with tetramethyl benzidine/tungstate/H2O2, or by their characteristically pale and distended mitochondria with irregular cristae. Controls included model ultrathin sections containing high concentrations of various amino acids. Alternate serial sections were labelled with anti-glutamate and anti-gamma-aminobutyric acid (GABA). Data were analysed by computer-assisted image analysis. Density of glutamate labelling (gold particles per micron2) on whole retinal terminals was > 3 times higher than that on postsynaptic dendrites, and > 5 times higher than that on miscellaneous non-retinal non-glutamatergic terminals in the suprachiasmatic nucleus. The overall density of gold particles over retinal terminals was approximately 3 times higher than that over GABAergic terminals, in which glutamate-like immunoreactivity was mainly mitochondrial. Labelling of vesicles in retinal terminals was almost 5 times greater than the apparent labelling of vesicles in GABAergic terminals, underscoring the location of transmitter glutamate within synaptic vesicles in retinal terminals. In the retino-recipient region of the suprachiasmatic nucleus there was also a small population of non-retinal glutamatergic terminals. Their overall immunoreactivity was similar to or exceeded that of retinal terminals, but morphological features clearly distinguished between these two types of glutamate-containing terminals. The present results indicate that the vast majority of retinal terminals may use glutamate as a transmitter, in keeping with electrophysiological and neuropharmacological data from other sources. The possibility of cotransmitters within retinal terminals, suggested by the presence of dense-core vesicles among the glutamate-containing synaptic vesicles, has still to be addressed.

Morphological heterogeneity of the GABAergic network in the suprachiasmatic nucleus, the brain's circadian pacemaker

“…In a previous study (Castel et al 1993), using specificity test grids, this anti-GABA antiserum showed no or negligible cross-reaction with 5 other amino acids commonly found in brain tissue : glutamate, taurine, glycine, aspartate and glutamine. In some experiments, in order to abolish possible cross-reactivity with tissue glutamate, the anti-GABA antiserum was preabsorbed with 50-100 µ glutamate-glutaraldehyde conjugate overnight at 4 mC.…”

“…Note that boutons 2 and 3 also impinge synaptically on a small dendrite (d2). Bouton 2, which is probably retinal and glutamatergic (Castel et al 1993), makes an asymmetric synaptic contact with d2 ; the arrow indicates a thick postsynaptic density. Bouton 3, which is GABA-ir, forms a symmetric synapse with d2.…”

Connexin36 vs. connexin32, “miniature” neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

“…Electrophysiological evidence from single-unit recordings (Green and Gillette, 1982) and days-long multiple-unit recordings of SCN neurons in hypothalamic slices (Bouskila and Dudek, 1993;Gribkoff et al, 2003) revealed that neurons in the isolated SCN generate a coordinated circadian rhythm of loosely synchronized electrical activity regulated by oscillatory mechanisms involving transcription and translation of circadian genes (Hastings and Herzog, 2004). Excitatory glutamatergic inputs from the retina mediate entrainment of the circadian rhythm to the environmental light-dark cycle (Kim and Dudek, 1991;Castel et al, 1993;Mintz et al, 1999). Although the intact SCN has a "free-running" (i.e., not entrained by environmental cues) oscillatory pattern of electrical activity that approximates 24 h, isolated SCN neurons have rhythms that range from about 20 h to 28 h (Welsh et al, 1995;Honma et al, 1998Honma et al, , 2004.…”

Effects of 17β-Estradiol on Neuronal Cell Excitability and Neurotransmission in the Suprachiasmatic Nucleus of Rat

“…The oscillations of the SFF in the SCN have been verified by both in vitro and in vivo extracellular electrophysiological recordings (Inouye and Kawamura, 1979;Green and Gillette, 1982;Groos and Hendriks, 1982;Bos and Mirmiran, 1990). The phase of the rhythm is shown to be set by light signals which cause glutamate release from terminals of retinal ganglion neurons with projections to the ventral part of the SCN (Castel et al, 1993;Shirakawa and Moore, 1994). The output of the SCN is suggested to be translated into synchronization of the metabolic and hormonal activity and subsequently behavioral reactions of the animal to the external light-dark cycle (see Van Esseveldt et al, 2000).…”