Inhibitory, GABAergic Nerve Terminals Decrease at Sites of Focal Epilepsy

. Using an immunocytochemical method for the localization of the y­ aminobutyric acid (GABA) synthesizing enzyme, glutamic acid decarboxylase (GAD), we have observed GABAergic nerve terminals distributed throughout all lay­ ers of normal monkey sensorimotor cortex. These terminals displayed ultrastructural characteristics that suggested that they arose from aspinous and sparsely spinous stellate neurons. In monkeys (Macaca mulattaand M. fascicularis) made epileptic by cortical application r�falumina gel, a highly significant numerical decrease of GAD­ positive nerve terminals occurred at sites o f seizure foci indicating a functional loss of GABAergic inhibitory synapses. A loss of such inhibition at seizure foci could lead to epileptic activity of cortical pyramidal neurons. models

Abstract. Using an immunocytochemical method for the localization of the y aminobutyric acid (GABA) synthesizing enzyme, glutamic acid decarboxylase (GAD), we have observed GABAergic nerve terminals distributed throughout all lay ers of normal monkey sensorimotor cortex. These terminals displayed ultrastructural characteristics that suggested that they arose from aspinous and sparsely spinous stellate neurons. In monkeys (Macaca mulattaand M. fascicularis) made epileptic by cortical application r�falumina gel, a highly significant numerical decrease of GAD positive nerve terminals occurred at sites o f seizure foci indicating a functional loss of GABAergic inhibitory synapses. A loss of such inhibition at seizure foci could lead to epileptic activity of cortical pyramidal neurons.
Epilepsy is a neurological disorder characterized by intermittent, general ized seizures involving motor and sen sory systems. Most epilepsies in humans are caused by tumors or by trauma to a brain region as a result of cranial in juries, including those produced by birtr canal obstructions (/). Human epilepsy has not been studied in as much detail as disorders in experimental animals pro duced by the application of various Macaca mulatta agents that initiate seizure activity; for example, alumina gel, cobalt, and pen icillin (2). Since these experimentally produced epileptic foci develop repro ducibly they have provided models for the study of human focal epilepsy.
Several factors, including glial hyper trophy, ischemia, and increased concen trations of acetylcholine, have been sug gested as being involved in the onset of seizures (3). Another such factor, and one that is the concern of this report, in volves the inhibitory neurotransmitter y aminobutyric acid (GABA). It has been suggested that a reduction of this inhib itory substance could be responsible for seizure activity because results from bio chemical studies have shown decreases in both GABA (4) and its synthesizing enzyme, glutamic acid decarboxylase (GAD) (5), at seizure foci.
In a recent immunocytochemical study, GAD has been localized within the axon terminals of aspinous and sparse ly spinous stellate neurons in the rat cerebral cortex (6). Since GAD has been found in a number of other brain regions within neurons that have been identified as GABAergic (7), it is probable that the presence of GAD is indicative of neurons that use GABA as a neurotransmitter. Therefore, the localization of GAD with in aspinous and sparsely spinous stellate neurons, together with evidence from physiological and pharmacological stud ies (8), strongly suggests that these neu rons are responsible for GABA-mediated inhibition in the cerebral cortex. Since these neurons are found in every cortical layer and project numerous axon termi nals to pyramidal cell somata, they could exert a powerful inhibitory effect on cor tical projection neurons. Furthermore, a decrease in the number of these inhib itory axon terminals could lead to sei zure activity of pyramidal neurons. To test this possibility, we compared the  these analyses indicate a highly signifi cant decrease in the number of GABA ergic axon terminals at sites of seizure foci. The decreased number of GAD-posi tive terminals in our immunocytochemi cal preparations of epileptic monkey cor tex could be explained by an alumina gel produced loss of antigenicity of GAD molecules. However, this is unlikely since most of the alumina is located in macrophages with slight amounts in as trocytes (/6). Furthermore, not all of the GAD-positive terminals are lost at seiz ure foci, and the staining of the remain ing terminals indicates that the antigenic ity of GAD is not affected by the alumina gel. In addition, a differential loss of anti genicity caused by differences in the dif fusion of alumina from the application site is unlikely since, in monkeys with subarachnoid injections, the deep cortic al layers display similar decreases of GAD-positive terminals to those ob served in the superficial layers directly subjacent to the alumina gel. Therefore, the loss of immunocytochemically de tectable GAD from cortical axon termi nals indicates an actual loss of GAD molecules and this could be due either to a severe impairment of GAD synthesis, or to the degeneration of GABAergic so mata or their axon terminals, or both.
central nervous system (18). Thus, our results in combination with those of the other studies cited in this report sup port a hypothesis that a loss of func tional GABAergic neurons leads to focal epilepsy.
Although previous biochemical data have indicated decreased GAD activities at seizure foci (5), our results extend this finding and show a numerical decrease of GAD-containing axon terminals. Wheth er these terminals actually degenerate or merely lose immunocytochemically de tectable GAD is not known. However, a degeneration of GAD-containing termi nals is suggested by the results of ultra structural studies that show a decreased number of presumed inhibitory, symmet ric synaptic junctions with somata and dendritic shafts of cortical neurons at seizure foci (/7). In either event, a func tional loss of GABAergic cortical neu rons would occur. Our experimental preparations indicate that the magnitude of this loss is significant and could be ex pected to reduce the inhibitory synaptic control of pyramidal neurons, thus lead ing to a hypersensitivity of these cells to normal excitatory synaptic inputs (4,5). The reason for this loss of GABAergic terminals at seizure foci is unknown, but it is possible that aspinous stellate neu rons may be highly susceptible to altera tions induced by alumina treatments. Further support for a GABAergic in volvement in epilepsy is derived from pharmacological studies that show that certain convulsant and anticonvulsant drugs act at GABAergic synapses in the