Activation of GABAA Receptors in Subthalamic Neurons In Vitro: Properties of Native Receptors and Inhibition Mechanisms

Abstract

The subthalamic nucleus (STN) influences the output of the basal ganglia, thereby interfering with motor behavior. The main inputs to the STN are GABAergic. We characterized the GABAA receptors expressed in the STN and investigated the response of subthalamic neurons to the activation of GABAA receptors. Cell-attached and whole cell recordings were made from rat brain slices using the patch-clamp technique. The newly identified ε subunit confers atypical pharmacological properties on recombinant receptors, which are insensitive to barbiturates and benzodiazepines. We tested the hypothesis that native subthalamic GABAAreceptors contain ε proteins. Applications of increasing concentrations of muscimol, a selective GABAAagonist, induced Cl− and HCO[Formula: see text]currents with an EC50 of 5 μM. Currents induced by muscimol were fully blocked by the GABAAreceptor antagonists, bicuculline and picrotoxin. They were strongly potentiated by the barbiturate, pentobarbital (+190%), and by the benzodiazepines, diazepam (+197%) and flunitrazepam (+199%). Spontaneous inhibitory postsynaptic currents were also significantly enhanced by flunitrazepam. Furthermore, immunohistological experiments with an ε subunit-specific antibody showed that the ε protein was not expressed within the STN. Native subthalamic GABAA receptors did not, therefore, display pharmacological or structural properties consistent with receptors comprising ε. Burst firing is a hallmark of Parkinson's disease. Half of the subthalamic neurons have the intrinsic capacity of switching from regular-firing to burst-firing mode when hyperpolarized by current injection. This raises the possibility that activation of GABAA receptors might trigger the switch. Statistical analysis of spiking activity established that 90% of intact neurons in vitro were in single-spike firing mode, whereas 10% were in burst-firing mode. Muscimol reversibly stopped recurrent electrical activity in all intact neurons. In neurons held in whole cell configuration, membrane potential hyperpolarized by −10 mV whilst input resistance decreased by 50%, indicating powerful membrane shunting. Muscimol never induced burst firing, even in neurons that exhibited the capacity of switching from regular- to burst-firing mode. These molecular and functional data indicate that native subthalamic GABAA receptors do not contain the ε protein and activation of GABAA receptors induces membrane shunting, which is essential for firing inhibition but prevents switching to burst-firing. They suggest that the STN, like many other parts of the brain, has the physiological and structural features of the widely expressed GABAA receptors consisting of αβγ subunits.