Imaging Neural Circuits Via Two-Photon-Excited Fluorescence And Second Harmonic Generation Microscopy
A functional neural circuit depends on multiple layers of complexity, from the cellular-level challenge of integrating synaptic inputs to the systems-level problem of deciphering spike patterns. A complete understanding of neural circuits cannot come from a single experimental approach, although laser-scanning microscopy methods promise new advances because optical emissions can potentially provide different kinds of information. As part of this Dissertation, I have developed nonlinear optical methods based on two-photon-excited fluorescence and second harmonic generation that reveal various structural and functional aspects of neural circuits. Microtubules are the "molecular highways" within neurons and act as the substrate for intracellular trafficking. The polarity of microtubules determines the directions and speeds of cargo-carrying molecular motors. Using second harmonic generation as an optical probe of non-centrosymmetric structures, polarized microtubule arrays were observed within axons and mature apical dendrites, but not within basal and oblique dendrites. This microtubule organization in native brain tissues is age-dependent and different from what is known for dissociated neuronal cultures. In addition to second harmonic generation, some biological molecules can emit autofluorescence. In transgenic Alzheimer's disease mouse models, both types of intrinsic optical emissions were observed at senile plaques. The utility of these spectrally broad but relatively weak signals was demonstrated by investigating the morphology of polarized microtubule arrays near senile plaques. The appearance, length, and density of neuronal microtubule arrays are normal even in the presence of pathological lesions, suggesting that apical dendrites may be more tolerant to pathological mechanisms compared to smaller neurites. Recording calcium transients with fluorescent indicator dyes is an indirect method to simultaneously measure spiking activity in many neurons. Applying two-photon calcium imaging to neonatal mouse spinal cords, one type of excitatory interneuron, defined by the transcription factor Hb9, was found to be fictive locomotion-related. However, these interneurons are not likely to be the rhythm generating kernel for the spinal locomotor circuit because the onset of their activity lags behind the onset of the ipsilateral motor output. Moreover, these interneurons show sparse, uncoordinated activity and may be important for initiating episodic fictive locomotor activity because they are most active immediately following the applied stimuli.
dissertation or thesis