Dendritic spines, which are literally tiny spines found in numerous quantities on the branched projections of neurons (dendrites), are known to be inputs for almost all excitatory electrical signals into neurons, which in turn can stimulate other neurons. Last year, the laboratories of Kenneth B. Eisenthal and of Rafael Yuste, both at Columbia University in New York, discovered that dendritic spines are not merely passive structures but that they actually can affect the electrical signal by acting as resistance filters, while some send no electrical signals at all, for reasons yet to be explored. Because these spines are only about 1 μm in size, they cannot accommodate an electrode, the traditional tool for measuring electrical signals in neurons. Eisenthal said that the investigators use second-harmonic-generation imaging to monitor the electrical potential in dendritic spines because when other imaging methods, such as fluorescence imaging, are used, the fluid medium of neurons overwhelms the signal from the membrane of the dendritic spines, where voltage signals are generated. In contrast, second-harmonic-generation imaging measures light scattering only from regions without symmetry, such as interfaces and surfaces. Therefore, the researchers could look specifically at the surface membrane of the dendritic spines. One can see dendritic spines in this second-harmonic-generation image of a dendrite from the neuron shown in the preceding figure.However, second-harmonic-generation chromophores yield signals with low intensities, resulting in poor-quality images. The poor-quality images limit the potential of the technique to reveal biological truths, such as why some dendritic spines modulate electrical signals where-as others do not deliver a signal. To improve second-harmonic-generation chromophores, one must understand the mechanism by which they generate a second harmonic.Thus, the researchers recently explored the mechanism of FM 4-64, the chromophore that they used to study dendritic spine membranes. They showed that the FM 4-64 chromophore produced a second harmonic signal that corresponded linearly to the changing voltage of the dendritic spine membranes, though the studies did not reveal why. The scientists hypothesized that the second harmonic light produced in response to changing voltage was in response to an electro-optic mechanism. However, it also was possible that the voltage changes could alter the orientation of the chromophore or cause the chromophore to exit the membrane and thus yield a voltage-dependent second harmonic signal. Finally, the voltage-dependent signal could be generated also by the Stark effect — the splitting or shifting of spectral lines as the result of an electric field.‘Picture perfect’ mechanismAs reported in the September issue of Biophysical Journal, the researchers studied the second-harmonic-generation mechanism using a two-photon laser scanning confocal microscope from Olympus America Inc. of Center Valley, Pa., with many postfactory modifications. The system contained a powerful femtosecond laser because the intensity of the second harmonic scales as the square of the intensity of incoming light, yet the pulses cannot be too powerful or they will damage the neurons. The scientists collected the second harmonic signals with a Hamamatsu photomultiplier tube. To determine the orientation of the chromophore, they performed polarization measurements by varying the polarization of the incident light with a quartz zero-order half-wave plate from Newport Corp. of Irvine, Calif. The researchers performed measurements within 1 ms, before the chromophore could exit the dendritic membrane. These experiments were carried out by Jiang Jiang, a postdoctoral associate in Yuste’s laboratory.Researchers captured this second-harmonic-generation image of a mouse neuron injected with the chromophore FM 4-64. The outline of the plasma membrane and the dendrites are visible, and the second harmonic signal intensity is directly proportional to membrane potential.The researchers discovered that the orientation of the chromophore did not change in response to electrical signals, even those as high as 90 mV. Also, the second-harmonic-generation response to changes in voltage occurred in less than 1 ms, so they knew that the chromophore did not exit the membrane. Finally, they concluded that second-harmonic generation did not occur as a result of the Stark effect because it did not have a strong wavelength dependency past the absorption peak. “FM 4-64 has a picture-perfect electro-optic mechanism,” Yuste said. Thus, increasing the polarity of the chromophore is key to increasing the second harmonic signal, Eisenthal said. He also said that they plan to attach metal nanoparticles to the chromophores to increase the second harmonic signal.