Establishment And Application Of Dendritic Patch-clamp Recording

The patch clamp technique is a laboratory technique in electrophysiology that allows the study of ion channels in cells. It provides a direct way to understand the gating kinetic characteristics of ion channels and the permeability of biomembrane. Many neuroscientists used this technique to explore the distribution and the function of ion channels in neuron. Patch-clamp recording is mostly performed on the soma of neuron. However, although dendrites are important for neuronal function, few studies tried to make patch-clamp recording from dendrites because of their tiny diameter. We know that dendrites receive the majority of a neuron’s synaptic inputs, and display a range of excitable properties, which are critical for synaptic intergration and shaping synaptic plasticity. So, direct dendritic patch-clamp recording would provide a new opportunity for further understanding of dendritic excitability.After our successful patch-clamp recording from soma, we attempted to record from dendrites. We performed on the healthy dendrites of CA1pyramidal neurons in hippocampal slices by the use of differential interference contrast (DIC) optics. Although the basic technique is similar to that used for somatic patching, dendritic recording needs more attention than the latter. After many experiments, we described refinements and optimizations of slice quality, setup stability, microscope optics and electrode approaches that were required for maximizing the success rate for dendritic recordings.We recorded from soma and dendrite simultaneously so as to study the passive and active properties of dendrites. The same excitatory postsynaptic current (EPSC)-shape current injection was given in the different domains of apical dendrite respectively. The result indicated that the site of current injection was farther from the soma the amplitude of dendritic excitatory postsynaptic potential (EPSP) was larger. Conversely, the amplitude of EPSP simultaneously recorded from soma was smaller, but the Half-width and the Rise time of somatic EPSP were longer. From these results we can conclude that EPSP generated in the distal apical dendrite was larger than that generated close to soma induced by the same current injection, and the amplitude of EPSP was attenuated whereas the duration was prolonged gradually when it propagated from generated sites to the soma. We applied glass electrode to stimulate apical dendrites in the stratum radiatum (SR), recording from soma and apical dendrite simultaneously, and obtained the same result. Based on these results we can conclude that the EPSP efficacy depends on the synapse location.Next, we gave somatic EPSC-shape current injection and patched soma and apical dendrites simultaneously. We found that when EPSP propagated from soma to dendritic recording sites its amplitude attenuated as well.We encountered many troubles when we endeavoured to make patch-clamp recording from basal dendrites of CA1pyramidal neurons because of the extremely tiny diameter. This property of basal dendrites increased the difficulty during patching them. Eventually, analyzing the possible reasons of failure and increasing practices made us obtain basal dendritic recordings.Once we become proficient enough to reliably obtain high-quality seals in dendritic recordings we plan to explore the integration mechanism of temporally correlated EPSPs initiated at different dendrites. Glass and metal electrodes were used to excite apical dendrites in the SR and basal dendrites in the stratum oriens (SO). We frist applied pairs of stimuli in which the SO stimulis preceded the SR stimulis by10 ms (defined as a-10ms interval). Pairing of small subthreshold SR and SO synaptic inputs at this temporal offset for6s at10Hz induced a large and sustained potentiation (LTP). However, at+10ms interval, we obtained a long-term depression (LTD). We decide to investigate the integration way of apical dendritic EPSP and basal dendritic EPSP drawing assistance from simultaneous recording from apical dendrites and soma as well as simultaneous recording from soma and basal dendrites.