Glycine and glycine receptor immunoreactivity in brain and spinal cord

Abstract

To study the distribution of glycine immunoreactive neurons in the spinal cord and brain, antisera were raised against glycine conjugated to protein carriers. High-titer rabbit glycine antiserum was purified by affinity chromatography. Testing against other amino acids and peptides with immuno dot blots and ELISA assays showed little apparent cross-reaction with glutamate, aspartate, glutamine, taurine, and 17 other amino acids and related compounds. Similarly, the antiserum showed little apparent recognition of glycine when glycine was incorporated into peptides. A slight cross-reactivity with GABA, beta- alanine, and cysteine was found. Immunocytochemical labeling of tissue sections could be blocked with glycine conjugated to a heterologous carrier protein but not by other amino acids conjugated to that protein. Immunocytochemistry at the light microscope level with immunofluorescence and silver-intensified colloidal gold revealed a wide distribution of glycine-like immunoreactivity throughout all laminae of the rat spinal cord and in all segments studied from the cervical, thoracic, lumbar, and sacral cord. Immunoreactive boutons were found terminating on both cell bodies and on dendrites. Ultrastructural analysis with postembedding colloidal gold immunocytochemistry demonstrated large numbers of immunoreactive boutons making symmetrical type synapses with neuronal perikarya, including motor neurons, and with proximal and distal dendrites. Presynaptic glycine immunoreactive boutons were found in both ventral and dorsal horn. Immunoreactivity was concentrated over regions rich in vesicles, and over mitochondria in immunoreactive boutons, but not over mitochondria in postsynaptic dendrites. Glycine-immunoreactive perikarya were identified both in the dorsal horn and in the ventral horn. Myelinated and unmyelinated glycine-immunoreactive axons were noted both in the gray and white matter of the cord. The density of immunoreactive axons varied in the white matter, with the greatest number of immunoreactive axons found in the white matter adjacent to the gray matter in lateral and ventral white. Significantly fewer immunoreactive axons were found in the white matter of the dorsal columns. Myelin sheaths around axons were unlabeled. The distribution of glycine-immunoreactive boutons correlated well with the distribution of glycine receptor immunoreactivity on postsynaptic elements of the spinal cord, tested with different monoclonal antisera against strychnine-purified glycine receptor. Glycine receptor immunoreactivity was found throughout the gray matter of both rat and primate.

Glycine-Containing Axon Terminals on Large Ventral Horn Cells Including Identified .ALPHA.-Motoneurons in the Spinal Cord of the Chicken.

“…Most glycine-LI synapses were symmetrical, but in a few instances asymmetrical synapses were found, which was in agreement with the situation in the rat [21]. In the dorsal horn, however, Todd [18] reported only the symmetrical type.…”

“…In the dorsal horn, however, Todd [18] reported only the symmetrical type. The synaptic vesicle shape in glycine-LI presynaptic boutons was of the pleomorphic or elongated type, as reported previously [14,16,21]. As for the correlation between vesicle shape and the function of the chemical synapse, it has been suggested that axon terminals containing round vesicles are excitatory and those containing elongated vesicles are inhibitory [20].…”

“…Electrophoretically administered glycine hyperpolarizes spinal motoneurons, and strychnine reversibly blocks that action [7,22]. Large ventral horn cells were demonstrated to receive glycinergic inputs by immunocytochemical technique [15,21] and by uptake of [3H]-glycine [12, 14,16] in the rat and cat. Glycine receptor has been demonstrated to be associated with large ventral horn cells [2,19,21].…”

“…Large ventral horn cells were demonstrated to receive glycinergic inputs by immunocytochemical technique [15,21] and by uptake of [3H]-glycine [12, 14,16] in the rat and cat. Glycine receptor has been demonstrated to be associated with large ventral horn cells [2,19,21]. Recently, glycine has been demonstrated to exist in Renshaw cells and Ia inhibitory neurons which play recurrent and reciprocal inhibitory roles on motoneurons [10].…”

Neurotransmitter signaling in white matter

“…High concentrations of neurotransmitter such as GABA and glycine are also present in a sub-population of mature WM axons in several species (Carlton et al, 1996;Davanger et al, 1991;Rogers and Pow, 1995;Todd and Sullivan, 1990;van den Pol and Gorcs, 1988;Wilson et al, 1996). Block of GABA uptake mimics the effects of GABA upon axon conduction in the neonatal rat optic nerve (Sakatani et al, 1991), while block of catecholamine uptake mimics the effect of nor-adrenaline (Nikolaeva et al, 2009); observations consistent with tonic operation of functional neurotransmitter uptake in the tissue.…”