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Tau is a microtubule associated protein that is localized to the axon in neurons. During pathological conditions, including frontotemporal dementia (FTD), a shift in tau isoforms occurs that leads to enhanced expression of a form of tau with four (rather than three) microtubule binding repeats; this has been postulated to alter microtubule structure. Second harmonic generation (SHG) is a technique that allows the visualization of intact microtubules in axons of living neurons without the need for labeling or fixing. We examined how the presence of exogenous tau influences SHG in living neurons. Our results show that the presence of tau significantly enhances SHG, specifically in neuronal axons, despite the presence of tau throughout the entire cell. Our data also suggest that the presence or absence of the fourth microtubule binding repeat does not significantly alter tau’s ability to enhance SHG. These results provide evidence that SHG is a useful, noninvasive tool to study tau-microtubule interactions in axons; further, it appears that tau overexpression, rather than specific isoforms, is the major contributor to tau-induced changes in axonal microtubule SHG signal. [ABSTRACT FROM AUTHOR]

Real-time investigation of cerebral blood flow (CBF), and oxy- and deoxyhemoglobin concentration (HbO, HbR) dynamics has been difficult until recently due to limited spatial and temporal resolution of techniques like laser Doppler flowmetry and magnetic resonance imaging (MRI). The combination of laser speckle flowmetry (LSF) and multispectral reflectance imaging (MSRI) yields high-resolution spatiotemporal maps of hemodynamic and metabolic changes in response to functional cortical activation. During acute focal cerebral ischemia, changes in HbO and HbR are much larger than in functional activation, resulting in the failure of the Beer-Lambert approximation to yield accurate results. We describe the use of simultaneous LSF and MSRI, using a nonlinear Monte Carlo fitting technique, to record rapid changes in CBF, HbO, HbR, and cerebral metabolic rate of oxygen (CMRO2) during acute focal cerebral ischemia induced by distal middle cerebral artery occlusion (dMCAO) and reperfusion. This technique captures CBF and CMRO2 changes during hemodynamic and metabolic events with high temporal and spatial resolution through the intact skull and demonstrates the utility of simultaneous LSF and MSRI in mouse models of cerebrovascular disease. [ABSTRACT FROM AUTHOR]

The analysis of fluorescence lifetime imaging microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles. [ABSTRACT FROM AUTHOR]

The Tg2576 transgenic mouse model of human cerebral amyloid angiopathy is characterized by age-dependent cerebrovascular deposition of amyloid-β (Aβ) starting from 9 months of age and progressively worsening to involve most pial arterioles by 18 months; soluble Aβ levels are elevated long before vascular deposition takes place in this model. It has been suggested that elevated soluble Aβ levels alone are sufficient to impair cerebral blood flow (CBF) regulation thereby contributing to the early progression of Alzheimers disease. Using laser speckle flowmetry through an intact skull, we studied the impact of elevated soluble Aβ levels and vascular Aβ deposition on a wide range of CBF responses to evaluate vasodilation and vasoconstriction in young or aged Tg2576 mice. Nineteen-month-old Tg2576 with severe vascular Aβ deposits showed an attenuated hyperaemic response during hypercapnia and whisker stimulation compared to wild-type littermates. The anticipated increase in CBF due to isoflurane anaesthesia was also suppressed, as were the typical hypoperfusion responses during cortical spreading depression and α-chloralose anaesthesia. The responses of 8-month-old Tg2576 with elevated soluble Aβ levels, but without vascular Aβ deposition, did not differ from age-matched controls. In conclusion, our data suggest that vascular Aβ deposition is associated with impaired vasodilator as well as vasoconstrictor responses to a wide range of stimuli. These responses do not differ from controls when studied non-invasively prior to vascular Aβ deposition, thus challenging the view that elevated soluble Aβ levels are sufficient to cause cerebrovascular dysfunction. [ABSTRACT FROM AUTHOR]