Double labeling immunoelectron microscopic study on the synaptic connections between glutamic acid neurons and GABA neurons in the hippocampus of rats

Abstract

In order to explore the roles of different neurotransmitters in epileptic pathogenesis, the synaptic connections between glutamic acid (Glu) neurons and GABA neurons in normal rat hippocampus were studied by pre-embedding double labeling immunoelectron microscopy. The GABA immunoreaction was first demonstrated by chromogen DAB, then the Glu immunoreaction was demonstrated by molybdic acid-TMB method. After being stabilized by DAB-cobalt chloride, the sections were processed for electron microscopic embedding. Under electron microscope, there were many Glu immunoreaction-positive neurons in the pyramidal layer of hippocampal CA₁ area and some GABA immunoreaction-positive neurons with pyramidal or polygonal perikarya in the pyramidal, polymorphic and radiant layer of CA₁ area. There were also symmetric dendro-axonic synapses formed by GABA-positive dendrites and Glu-positive axons in the polymorphic layer and symmetric axo-dendritic synapses formed by GABA-positive axons and Glu-positive dendrites in the radiant layer. In addition, there were symmetric autoregulatory axo-dendritic synapses between Glu-positive axons and dendrites and autoregulatory axo-axonic synapses (both symmetric and asymmetric) between GABA-positive axons. Above mentioned results, for the first time, showed that there were complex synaptic regulatory relationships between excitatory Glu neurons and inhibitory GABA neurons in the hippocampal CA₁ area, thereby, providing ultrastructural evidence for different neurotransmitters participating in epileptic pathogenesis.

Keywords

hippocampus / glutamic acid / gama-aminobutyric acid / synapse / immunoelectron microscopy

References

[1] UrbanL, AitkenP G, CrainB J, et al.. Correlation between function and structure in “Epileptic” human hippocampal tissue maintained in vitro. Epilepsia, 1993, 34(1): 54-54

[2] TaskerJ G, DudekF E. Electrophysiology of GABA-mediated synaptic transmission and possible roles in epilepsy. Neurochem Res, 1991, 16(3): 251-251

[3] ChapmanA, MelrumB. Excitatory amino acids in epilepsy and novel anti-epileptic drug. Epilepsy Res, 1991, 3(1): 39

[4] NorgrenR B, LehmanM N. A double-label pre-embedding immunoperoxidase technique for electron microscopy using diaminobenzidine and tetramethyl-benzidine as markers. J Histochem Cytochem, 1989, 37(8): 1283-1283

[5] NT SP 1997, 7(1):43

[6] WalaasI. EmsonPC. The Hippocampus. Chemical Neuroanatomy, 1983WuhanRaven Press377-358

[7] GamraniH, OntenienB, SeguelaP, et al.. Gama-aminobutyric acid immunreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies. Brain Res, 1986, 364(1): 30-30

[8] GABA 1994, 25 (4):345

[9] MaloufA T, RobbinsC A, SchwartzkroinP A. Epileptiform activity in hippocampal slice cultured with normal inhibitory synaptic drive. Neurosci Lett, 1990, 108(1–2): 76-76

[10] SP 1991, 22(4):357

[11] ThindK K, GoldsmithP C. Infundibular gonadotropin-releasing hormone neurons are inhibited by direct opioid and autoregulatory synapses in juvenile monkeys. Neuroendocrinology, 1988, 12(2): 317-317