To Study The Role Of External Globus Pallidus In The Production Of Abnormal Involuntary Movements

The basal ganglia are a group of subcortical nuclei. The main components of the basal ganglia are the striatum (caudate nucleus and putamen), external globus pallidus (GPe), internal globus pallidus (GPi)-substantia nigra pars reticulate (SNr), substantia nigra pars compacta (SNc) and the subthalamic nucleus (STn). The role of the basal ganglia in motor control is debated. A considerable advance was made in understanding the role of the basal ganglia with the development of what has come to be commonly called the direct pathway and indirect pathway model of basal ganglia. However, the direct pathway and indirect pathway models of basal ganglia are still incapable to explain all the neural circuitry of all the movement disorders which are associated with the basal ganglia dysfunction. The role of the specific neuron subtype of the basal ganglia in movement disorders needs to be further studied.GPi is the primary output nuclei of the BG. Most of the movement disorders are associated with the dysfunction of GPi. The recent literature confirms the efficacy of high-frequency electrical stimulation of the GPi for parkinson disease, primary dystonia, huntington disease. But the function of GPe is not clear. The GPe is a component of the indirect pathway. The GPe receives massive GABAergic afferent fibers from the striatum and glutamatergic afferent fibers from the STn. The chorea (hyperkinesia) characteristic of the early stage of the Huntington Disease (HD) appears to stem from preferential loss of ENK+striatal neurons. Due to loss of the inhibitory ENK positive striatal neurons projecting to the GPe, the GPe activity is increased. Excess GPe activity may be the cause of chorea in HD. Previous experiments have shown that abnormal involuntary movements could be induced by using microinjections of bicuculline into the GPe in primates. In a word, excess GPe activity may be the cause of abnormal involuntary movements. The GPe may be another good therapy target for movement disorders besides the GPi.Traditional methods in neural circuits study include lesion animal model, functional image study, electrical stimulation, microinjection of drug. All these methods could not manipulate individual components of the brain with high-temporal resolution. Only in recent years, the emergence of optogenetics makes it possible. Neurons may be controlled by optogenetics for fast, specific excitation or inhibition in freely moving mammals. For example, Channelrhodopsins (ChR2) conduct cations and depolarize neurons upon blue light illumination, while natronomonas pharaonis halorhodopsins (NpHR) conduct chloride ions into the cytoplasm upon yellow light illumination. Here we can use the optogenetic tools to study the function of GPe GABAergic neurons in motor control and the neural circuitry of the basal ganglia.Part1The effects of direct optical stimulation of local GPe GABAergic neuronsThe majority of GPe neurons are projection neurons that contain glutamate decarboxylase (GAD). VGAT-ChR2-EYFP transgenic mice are used for precisely controlling action potential firing of GABAergic neurons using blue light. A fiber guide was inserted to the right GPe of the VGAT-ChR2-EYFP transgenic mouse. After illumination of the right GPe by blue light, the mouse developed twisted postures of the neck and the left arm abnormal posture resembling dystonia, repetitive involuntary movements (licking and chewing) and rotation. All the abnormal involuntary movements disappeared after laser off. EEG recordings from the motor cortex during the optical stimulation did not show any abnormal activity. So these involunrary movements were not caused by seizures. We first validated that stimulation of GPe GABAergic neurons could induce dystonia like behavior and involuntary movements. We examined the expression of the immediate early gene, c-Fos, a biomarker of active neurons, in the basal ganglia loop (GPe, GPi, STn, M1). Our findings suggest the neural circuitry of the abnormal involuntary movements is related with excess GPe activity, reduced GPi activity, and excess motor cortex activity.Part2The effects of optical stimulation of excitatory afferent axons in GPeThe GPe receives massive GABAergic afferent fibers from the striatum and glutamatergic afferent fibers from the STn. The Thyl-ChR2-EYFP transgenic mice are used for precisely controlling action potential firing of excitatory projection neurons using blue light. We established that ChR2-EYFP was expressed in the excitatory afferent axons in GPe, but not cell bodies of GPe. A fiber guide was inserted to the right GPe of the Thyl-ChR2-EYFP transgenic mouse. After illumination of the right GPe by blue light, the mouse developed the similar phenotype as VGAT-ChR2-EYFP transgenic mouse in the first part, such as twisted postures of the neck and the left arm spasm resembling dystonia and rotation. EEG recordings from the motor cortex during the optical stimulation did not show any abnormal activity. So these abnormal involuntary movements were not caused by seizures. The results indicated that increasing the activity of excitatory afferent axons in GPe could also induce abnormal involuntary movements. The increase of activity of afferent axons in GPe may be the neural substrates in some movement disorders. The excess GPe activity play a critical role in producing abnormal involuntary movements.Part3The effects of optical stimulation of specific glutamatergic afferent fibers from STn in GPeThe GPe receives glutamatergic afferent fibers from the STN. In the second part we cannot establish whether the glutamatergic afferent fibers stimulated in GPe come from the STn. In this part, we delivered AAV carrying ChR2-mCherry under the CaMKIIa promoter to the right STn of the mice. ChR2-mCherry expression was specific to STn excitatory neuron cell bodies and precesses. ChR2-mCherry was expressed in the afferent fibers in the GPe. A fiber guide was inserted to the right GPe of this mouse. After illumination of the right GPe by blue light, the mouse developed the similar phenotype as VGAT-ChR2-EYFP transgenic mouse and Thyl-ChR2-EYFP transgenic mouse in the first and second part, such as twisted postures of the neck and rotation. The results shown that optical stimulation of specific glutamatergic afferent fibers in GPe, which come from STn, would cause abnormal involuntary movements. So the abnormal activity of this specific neuron pathway (glutamatergic projection neurons from STn to GPe) may be involved in the neural circuitry mechanism of abnormal involuntary movements related movement disorders. We also emphasized the important role of excess GPe activity in producing abnormal involuntary movements.Therefore, we first used optogenetic tools to reveal that excess GPe GABAergic neurons activity may be the cause of abnormal involuntary movements. This may be the neural substrates of abnormal involuntary movements related movement disorders. The increasing activity of specific glutamatergic afferent fibers in GPe, which come from STn, may be involved in the neural circuitry mechanism of abnormal involuntary movements related movement disorders. Excess GPe activity, reduced GPi activity and excess motor cortex activity may be the neural circuitry mechanism of the abnormal involuntary movements. The results provided an important clue to understand the neural circuitry mechanism of abnormal involuntary movements related movement disorders. GPe may be another good therapy target for abnormal involuntary movements related movement disorders.