Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons (original) (raw)

Journal of Visualized Experiments

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites. Video Link The video component of this article can be found at http://www.jove.com/video/54601/ 3 , the optics 4 and refinement of methods for slice preparation 5 during the last decades have enabled recordings from very thin (0.7-3 µm Ø) dendrites 6,7. These methods were, and are, still largely used to examine the excitability of dendrites in a variety of neurons 8. Direct dendritic recordings are essential to determine the distribution 9-19 and differences in the functional properties 20-22 of ion channels in distinct neuronal compartments. These data are the necessary complement of ion channel distributions detected with immunohistochemistry combined to light and electron microscopy 23,24. Dual somatodendritic recordings have been implemented to explore the propagation of action potentials 9,13-15,21,22,25-27 and spreading of synaptic potentials 13,16,18 along the somatodendritic domain of neurons, obtain detailed passive cable models 28-30 and investigate the temporal resolution of neuronal integration 31. The substantia nigra (SN) is a region located in the midbrain involved in several functions such as the control of movement, the coding of reward and habitual behaviors. The decrease of dopamine due to the specific loss of dopaminergic (DA) neurons in the SN is associated with the motor disturbances observed in patients suffering from Parkinson's disease 32. The nigral circuit is composed of two main cell types: dopaminergic and GABAergic neurons. Interestingly, these neurons have several specific features that distinguish them from other neurons. The axon of a large proportion of DA neurons and some GABA neurons originates from a dendritic site indicating that the dendritic arbor is heterogeneous (axonbearing and axon-lacking dendrites) 25,26,33. The morphology of these neurons contrasts therefore with the typical organization of neurons in which the information transfer follows the law of dynamic polarization emitted by Cajal: starting from dendrites, to soma and finally to axon 34. DA neurons are also known to release dopamine from their dendrites 35 , generate bursting activity 36 and NMDA-receptor plasticity 37. The dissection of these phenomena is elusive without direct recordings from the site where they are initiated. To gain insights into the relationship between the precise location and functional properties of ion channels and their role in the dendritic excitability and information transfer in nigral neurons, direct dendritic recordings are the method of choice. This report describes a detailed procedure that can be used to obtain single and dual patch-clamp recordings from dendrites of nigral neurons and the corresponding post hoc biocytin labeling. The basic principles for patching the somatic and the dendritic membrane are very similar. Practically however, recordings from dendritic sites require specific optimization in comparison to somatic recordings. Successful dendritic recordings rely on the quality of the slices, optimal adjustment of the optics, gentle approach of the patch pipette and stability of the recordings.