Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex

doi:10.1016/0306-4522(87)92968-X

Neuroscience

Volume 22, Issue 3, September 1987, Pages 897-912

https://doi.org/10.1016/0306-4522(87)92968-X Get rights and content

Abstract

Polyclonal antibodies were made in rabbits against glycine conjugated to bovine serum albumin with glutaraldehyde and were used for immunocytochemical studies in the cochlear nucleus and superior olivary nucleus of the guinea-pig. Antibodies selective for glycine were prepared by affinity chromatography. By dot-blot analysis this preparation showed a strong recognition of glycine conjugates and relatively little recognition of conjugates of most other amino acids tested. However, there was a significant reaction with conjugates of alanine and beta-alanine, and this cross-reaction could not be removed by affinity chromatography without eliminating the preparation's recognition of glycine. The affinity-purified preparation showed only a weak recognition of conjugates of gamma-aminobutyrate (GABA) which was detectable at high concentrations of primary antibody. Immunocytochemical studies showed several intensely staining cell bodies in the cochlear nucleus and superior olivary complex. Most immunoreactive cell bodies in the cochlear nucleus were in the dorsal cochlear nucleus, being present in both the superficial and deep layers. Scattered immunoreactive cells were present in the ventral cochlear nucleus. Intense staining of cell bodies was seen in the medial nucleus of the trapezoid body, and these cells appear to correspond to the principal cells of that nucleus. Punctate labelling, suggestive of immunoreactive presynaptic terminals, was also apparent, particularly in the ventral cochlear nucleus and lateral superior olive. In the ventral cochlear nucleus, immunoreactive puncta were found around unlabeled cell bodies, at times nearly covering the perimeter of the cell. A population of glycine-immunoreactive cell bodies in the superficial dorsal cochlear nucleus also labeled with anti-GABA antibodies as determined through double-labeling studies. However, glycine-positive cells in the deep dorsal cochlear nucleus were not labeled with anti-GABA antibodies, and some populations of GABA-positive cells in the superficial layers were not labeled with anti-glycine antibodies. In the hippocampus intense staining of cell bodies and puncta was seen with anti-GABA antibodies while essentially no staining was seen with anti-glycine antibodies.

These results suggest that anti-glycine antibodies can be useful for immunocytochemical identification of glycinergic neurons. From this study several populations of putative glycinergic neurons are identified in the auditory nuclei of the brain stem using these antibodies. Some populations of GABA-containing neurons also contain high levels of glycine or a related molecule.

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Hyperekplexia or startle disease is a dysfunction of inhibitory glycinergic neurotransmission characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. Although rare, this disorder can have serious consequences, including sudden infant death. One of the most frequent causes of hyperekplexia are mutations in the SLC6A5 gene, encoding the neuronal glycine transporter 2 (GlyT2), a key component of inhibitory glycinergic presynapses involved in synaptic glycine recycling though sodium and chloride-dependent co-transport. Most GlyT2 mutations detected so far are recessive, but two dominant missense mutations have been described. The detailed analysis of these mutations has revealed structural cues on the quaternary structure of GlyT2, and opens the possibility that novel selective pharmacochaperones have potential therapeutic effects in hyperekplexia.
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Citation Excerpt :
Both publications revealed that GlyRs are mainly expressed in the lower brain, including spinal cord and medulla oblongata. Interestingly, using anti-glycine antibodies, glycinergic neurons are also mainly detected in the lower brain [7,12]. Above data strongly indicate that glycinergic neurons, receptors, i.e. the inhibitory glycinergic system, are concentrated in the lower brain.
Glycine is an important amino acid in the central nervous system. Interestingly, the content of glycine is about 9 times higher in the spinal cord grey matter than in the telencephalon. And this kind of caudal to rostral gradient is never seen in any other neurotransmitters. However, the cause of this phenomenon remains unknown. In the present report, I, thus, postulate the following theory. Glycine has dual roles as a neurotransmitter, one is the agonist for inhibitory glycine receptors (GlyRs), and the other is a co-agonist for excitatory NMDA receptors (NMDARs). Inhibitory GlyRs are concentrated in the lower brain and the affinity of glycine to GlyRs is low, leading to the high content of glycine in the lower brain. In contrast, in the upper brain, there are little glycinergic neurons and the affinity of glycine to NMDARs is very high, leading to the low content of glycine in the forebrain. These different roles of glycine as a neurotransmitter between in the upper brain and in the lower brain make this steep caudal-rostral gradient in glycine content.
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Amino acids have important roles in the chemistry of the auditory system, including communication among neurons. There is much evidence for glutamate as a neurotransmitter from auditory nerve fibers to cochlear nucleus neurons. Previous studies in rodents have examined effects of removal of auditory nerve input by cochlear ablation on levels, uptake and release of glutamate in cochlear nucleus subdivisions, as well as on glutamate receptors. Effects have also been reported on uptake and release of γ-aminobutyrate (GABA) and glycine, two other amino acids strongly implicated in cochlear nucleus synaptic transmission. We mapped the effects of cochlear ablation on the levels of amino acids, including glutamate, GABA, glycine, aspartate, glutamine, taurine, serine, threonine, and arginine, in microscopic subregions of the rat cochlear nucleus. Submicrogram-size samples microdissected from freeze-dried brainstem sections were assayed for amino acid levels by high performance liquid chromatography. After cochlear ablation, glutamate and aspartate levels decreased by 2 days in regions receiving relatively dense innervation from the auditory nerve, whereas the levels of most other amino acids increased. The results are consistent with a close association of glutamate and aspartate with auditory nerve fibers and of other amino acids with other neurons and glia in the cochlear nucleus. A consistent decrease of GABA level in the lateral superior olive could be consistent with a role in some lateral olivocochlear neurons. The results are compared with those obtained with the same methods for the rat vestibular nerve root and nuclei after vestibular ganglionectomy.

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Glycine immunoreactivity localized in the cochlear nucleus and superior olivary complex

Abstract

Brain Res.

Brain Res.

Brain Res.

J. Neuropharmac.

Brain Res.

J. biol. Chem.

Brain Res.

Expl Neurol.

Brain Res.

Brain Res.

Brain Res.

Origins of axons in the cat's acoustic stria determined by injection of horseradish peroxidase into severed tracts

J. comp. Neurol.

Neurochemical evidence for glycine as a transmitter and a model for its intrasynaptosomal compartmentation

The distribution of glycine, GABA, glutamate and aspartate in rabbit spinal cord, cerebellum and hippocampus

J. Neurochem.

The neuronal architecture of the cochlear nucleus of the cat

J. comp. Neurol.

Pathways connecting the right and left cochlear nuclei

J. comp. Neurol.

The effects of inhibitory and excitatory amino-acid neurotransmitters on the response properties of brainstem auditory neurons

Amino acid transmitters in the mammalian central nervous system

Rev. Physiol.

Quantitative histochemical mapping of candidate transmitter amino acids in the cat cochlear nucleus

J. Histochem. Cytochem.

Purification and characterization of the glycine receptor of pig spinal cord

Biochemistry

Antisera to γ-aminobutyric acid. I. Production and characterization using a new model system

J. Histochem. Cytochem.

Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures

J. Histochem. Cytochem.

Anatomy of the eighth nerve. III. General plan of structure of the primary cochlear nuclei

Laryngoscope, St. Louis