Development of γ‐aminobutyric acid‐, glycine‐, and glutamate‐immunopositive boutons on rat jaw‐opening motoneurons

Abstract

Inhibitory and excitatory synaptic inputs onto trigeminal motoneurons play an important role in coordinating jaw movements. Previously, we reported that the phenotype of the inhibitory boutons apposing the somata of jaw-closing (JC) motoneurons changes from γ-aminobutyric acid (GABA)-positive (GABA+) to predominantly glycine-positive (Gly+) during development. In the present study, we investigated the development of inhibitory and excitatory boutons apposing antagonistic jaw-opening (JO) motoneurons (anterior digastric motoneurons) at postnatal day 2 (P2), P11, and P31 in the rat. JO motoneurons were retrogradely labeled with horseradish peroxidase. Postembedding immunogold staining with antisera against GABA, Gly, and glutamate (Glut) was performed and followed by quantitative ultrastructural analysis. The size of both small and large JO motoneurons increased during development. The number of excitatory (Glut+) and inhibitory (GABA+, Gly+, and GABA+/Gly+) boutons per JO motoneuron increased significantly from P2 to P11 and then remained unchanged until P31. The time course of inhibitory synapse formation differed between JO and JC motoneurons, whereas that of excitatory synapse formation was similar between the two neuronal populations. The fraction of GABA+ boutons decreased by 86% and the fraction of GABA+/Gly+ boutons increased by 200% from P11 to P31, suggesting a switch from GABA+ to GABA+/Gly+ phenotype. The fraction of Gly+ boutons remained unchanged. These results indicate that inhibitory synapses onto somata of JO motoneurons exhibit a developmental pattern distinct from that of synapses onto JC motoneurons, which may reflect distinctive maturation of oral motor system.

γ‐Aminobutyric acid‐, glycine‐, and glutamate‐immunopositive boutons on mesencephalic trigeminal neurons that innervate jaw‐closing muscle spindles in the rat: Ultrastructure and development

“…Test sections were incubated in the same drops of GABA, Gly, and Glut antisera as the tissue sections, and the conjugates in the test sections were selectively labeled. Also, the staining pattern of boutons apposed to somata of Vmes neurons was similar to that in our previous studies of jaw‐closing and jaw‐opening motoneurons in the rat and the cat (Bae et al,1999,2002; Shigenaga et al,2005; Paik et al,2007,2011,2012). Furthermore, omission or replacement of the primary antisera with normal rabbit serum or preadsorption of the diluted anti‐GABA serum with 200 μM GABA‐G, the antiglycine serum with 300 μM Gly‐G, and the antiglutamate serum with 300 μM Glut‐G abolished the specific immunostaining, confirming the selectivity of the antisera.…”

“…They were raised according to the procedure of Storm‐Mathiesen et al (1983), except that the amino acids were conjugated to bovine serum albumin by glutaraldehyde and formaldehyde instead of glutaraldehyde alone and extensively characterized (Ottersen,1989a; Broman et al,1993; Matsubara et al,1996; Takumi et al,1999). Their specificity was confirmed on test sections of “sandwiches” of rat brain macromolecule–glutaraldehyde complexes of different amino acids, including GABA, Gly, and Glut (Ottersen,1987; Zhang and Ottersen,1992; Bae et al,2000; Paik et al,2012). Test sections were incubated in the same drops of GABA, Gly, and Glut antisera as the tissue sections, and the conjugates in the test sections were selectively labeled.…”

Increase of glutamate in satellite glial cells of the trigeminal ganglion in a rat model of craniofacial neuropathic pain

“…Postembedding immunogold labeling for glutamate was performed according to the method described previously by our laboratory ( Paik et al, 2012 , 2021 ). Briefly, grids were treated for 6 min in 1% periodic acid, to etch the resin, and for 10 min in 9% sodium periodate, to remove the osmium tetroxide.…”

Glutamate Signaling, Associated With Satellite Glial Cells That Envelop Small Trigeminal Ganglion Neurons Is Highly Involved in the Neuropathic Pain

“…Postembedding immunogold staining for glutamate was performed according to the method described previously by us 41,42,43 . Brie y, grids were treated for 6 min in 1% periodic acid, to etch the resin, and for 10 min in 9% sodium periodate, to remove the osmium tetroxide.…”