Mapping the distribution of HCN1-subunit containing channels on the dendritic trees of trapezius motoneurons
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Voltage-dependent channels on the dendrites of motoneurons provide additional current that amplifies or dampens synaptic current en route to the soma. The specific consequences will depend on the density and distribution of the voltage-dependent channels. HCN channels generate a positive inward current in response to hyperpolarization. HCN channels are responsible for a resonance phenomenon in motoneurons where inputs of certain frequencies are preferentially amplified. Modelling studies indicate that this resonance behaviour only occurs if HCN channels are uniformly distributed on the dendritic tree. However, the distribution of HCN channels on the dendrites of motoneurons is unknown. Furthermore, current techniques for measuring channel density on dendrites suffer from methodological limitations that prevent sampling on a scale necessary to map the distribution of voltage-dependent channels across the dendritic tree. The goal of the present study is to develop a high throughput method for measuring the membrane-associated density of voltage-dependent channels using immunohistochemical, confocal and three-dimensional image analysis techniques. Secondly, the proximal to distal distribution of membrane-associated channels formed by the HCN1-subunit on the dendritic trees of trapezius motoneurons in the adult feline will be compared. Antidromically identified motoneurons innervating the trapezius muscle were intracellularly stained in order to visualize the entire dendritic tree. HCN1-subunit containing channels were labeled with a specific antibody. Dendritic segments (n=27 to 92) of ten trapezius motoneurons at different distances from the soma were acquired using confocal microscopy and rendered into a three-dimensional volume. The perimeter of the intracellular stain was used to define the membrane-cytoplasmic border. Analysis of the HCN1 immunoreactivitywas constrained to this perimeter. This technique provides a means of extracting membrane-associated HCN1 labeling and therefore the functional distribution of HCN1 channels. The density of membrane-associated HCN1 immunoreactvity across the dendritic tree was either uniform or increased with distance from the soma among the ten trapezius motoneurons. The increase in HCN1 density with distance from the soma was inversely related to the density of HCN1 on the soma and proximal dendrites. Lastly, the change in HCN1 density with distance from the soma was inversely related to the total input conductance of the motoneuron.