Ž . Developmental Brain Research 105 1998 219–225 Research report Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures Paul Honegger ) , Beatriz Pardo, Florianne Monnet-Tschudi Institute of Physiology, UniÕersity of Lausanne, CH-1005 Lausanne, Switzerland Accepted 7 October 1997 Abstract During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death andror rearrangement of neuronal connectivity. To study the Ž . effects of sustained inhibitory activity on developing inhibitory GABAergic neurons, three-dimensional primary cell cultures of fetal rat Ž . telencephalon were used. In relatively immature cultures, muscimol 10 mM , a GABA receptor agonist, induced a transient increase in A apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double Ž . strand breaks TUNEL labeling . Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABA receptor A Ž . Ž . Ž . blockers bicuculline 100 mM and picrotoxin 100 mM , by depolarizing potassium concentrations 30 mM KCl and by the L-type Ž . Ž . calcium channel activator BAY K8644 2 mM . As compared to the cholinergic marker choline acetyltransferase activity , glutamic acid Ž decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin 1 . Ž . Ž . Ž . mM , flunarizine 5 mM , MK 801 50 mM and propofol 40 mM . The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABA autoreceptor activation. q 1998 Elsevier Science B.V. A Keywords: Inhibitory interneuron; GABA; Apoptosis; Muscimol; Rat telencephalon; Aggregate cell culture 1. Introduction In the developing mammalian brain, programmed cell death occurs massively at the onset of neuronal activity w x 9,35 , suggesting that neuronal electrical activity is a powerful epigenetic modulator of neuronal viability. Inade- quate neuronal activity may affect neuronal viability w through the direct perturbation of ion homeostasis 3– x 6,11,12,24–26,38,39,42 , or indirectly through the restric- w x tion of neurotrophic support 14,37,40 . In many instances, differential responsiveness to activity-dependent modula- tors of neuronal survival has been observed between differ- w x ent neuronal subpopulations 5,27–31,39,43 . Therefore, the present work was undertaken to examine whether fundamental differences exist between excitatory and in- hibitory neurons in the vulnerability to agents that modu- ) Corresponding author. Institute of Physiology, University of Lau- sanne, 7, Rue du Bugnon, CH-1005 Lausanne, Switzerland. Fax: q41- 21-6925595; E-mail: paul.honegger@iphysiol.unil.ch late neuronal activity. In the cerebral cortex, the matura- tion of neuronal circuits is controlled by intrinsic neuronal w x activity 23 and the anatomical and functional rearrange- ment of neuronal connections appears to be the prevalent w x developmental strategy for projection neurons 34 . Much less is known about the fate of inhibitory interneurons. Inhibitory circuits, formed by a relatively small population of predominantly GABAergic neurons, have been shown w x to mature relatively late in development 2,16 . Therefore, they may be responsible for the balance of excitatory and inhibitory activities and thus for the final numerical match- ing between excitatory and inhibitory neuronal subpopula- tions. Activity-dependent signals reducing the number of inhibitory neurons would most likely affect those neurons lacking adequate excitatory input, implying that they should exhibit vulnerability to sustained hyperpolarization andror Ca 2q deprivation. To test this hypothesis, we examined the sensitivity of immature GABAergic neurons to inhibitors of neuronal activity, in particular to the GABA receptor A agonist muscimol, using serum-free aggregating brain cell cultures as a model. This culture system proved to be 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.