This is Part 4 of a 5-part series. See also Part 1, Part 2, Part 3, and Part 5.
Topic 3: New Vistas in Brain Imaging
27 December 2007. The technological limitations to studying Alzheimer disease remain major. For example, the ability to image changes in brain needs to move to a new level of analysis. In vivo-imaging, functional imaging, imaging of behaving animals, deep brain imaging, and 3D structural imaging of tissue are all priorities. On some of those, technical innovations in basic neuroscience have reached a point where they can now be imported into AD research. Below are three examples.
The field of in-vivo imaging of neural activity and neural circuitry advanced with the development of transgenic mice expressing channelrhodopsin-2 (ChR2). This membrane protein conducts non-selective currents in response to illumination with blue light. Several mouse lines now exist that express ChR2 fused to yellow fluorescent protein (YFP) selectively in the brain, such that in a given transgenic line, individual neurons fluoresce in a particular cortical area, the retina, motor nerves, etc. Both in brain slices, and in anesthetized mice, illuminating a respective area of cortex evokes light-activated action potentials from the ChR2-expressing neurons. Light intensities as low as 1 milliwatt per square millimeter depolarize the ChR2-expressing neurons, and milliseconds later produce recordable currents from those neurons. The operator can control the frequency of the neurons’ spike trains; that is, light pulses of up to 30 Hz produce corresponding spike trains of up to 30 Hz. Sustained illumination of up to several seconds generates sustained firing (Wang et al., 2007).
As a proof of principle that this technique is suitable to map circuits in the brain, an initial study applied it to trace the projections from mitral neurons in the olfactory bulb to the piriform cortex of the mouse brain, where olfactory stimuli undergo higher-order processing. A line of transgenic mice expressing ChR2 only in mitral neurons served this purpose. Illuminating olfactory bulb patches of varying diameter while recording from piriform cortex suggested strongly convergent mitral inputs onto piriform cortex neurons, rather than one-to-one connections (Arenkiel et al., 2007; Feng lab page).
This technique is robust enough to be applied to questions of changes in neural activity and circuits in AD research. One ongoing technical refinement involves the use of specific promoters to target ChR2 expression to specific subtypes of neuron (i.e., GABAergic, glutamatergic, etc.). Another involves combining ChR2 with additional genetically encoded calcium- or voltage-sensitive fluorescent proteins to improve the functional readout of exactly what is going on in neurons when they are stimulated. A current limitation of this technique is that, as with multiphoton imaging of AD mouse models, the light has to be delivered through a cranial window. Another is that light scattering and attenuation place any tissue deeper than 600 micrometers from the surface beyond reach. One option to overcome this limitation is to stereotactically implant a pinpoint source of light into transgenic mice, essentially attempting deep brain stimulation using light.
Besides seeing cellular dynamics in deeper layers, a second goal in brain imaging is to do so in freely moving animals, not sedated mice immobilized onto a microscope stage. Fluorescence microendoscopy uses minimally invasive micro-optic and fiberoptic probes from 1,000 down to 350 micrometers in diameter. The tiny objective lenses at their tips now exist as doublets or triplets and provide micron-scale lateral resolution. This technology can image capillary blood flow in the hippocampal CA1 region of mice (see prototype movie on Schnitzer lab page). It visualizes pyramidal neurons expressing YFP and could be applied to image transgenic mice of interest to AD research.
Two basic forms of microendoscopy are complementary to each other. Epifluorescence microendoscopy is similar to conventional epifluorescent microscopy, except it uses a long, thin probe to reach into the brain. The probe sends light into the brain and projects the image back onto a camera. It is simple and fast but prone to light scattering and does not generate true 3D images. In the two-photon laser-scanning form of microendoscopy, the focus of an ultrafast laser beam scans across the top face of a micro-optic probe, and the scan pattern is projected into the brain. The excited fluorescence passes back through the system and is captured by a photodetector that allows optical sectioning through 3D data stacks. This method allows deeper penetration into the brain, focal excitation, and is robust to scattering. Examples of high-quality imaging with this technology include pictures of the rows of outer and inner hair cells in the cochlea of live guinea pigs, excised human cochlea, as well as gentamycin-induced damage to those cells (Monfared et al., 2006).
More recent refinements to this technology have made possible long-term neuronal imaging in the same animal for up to a year. Guided tubes mounted on the heads of mice ensure that repeatedly inserted microendoscopy probes image the same hippocampal region, with the same 10 to 20 fluorescent neurons and dendrites in the visual field, day after day. As an initial experimental application, this technology has enabled the observation of tumor growth over time.
The long-standing goal of imaging the brain of freely moving rodents had an early breakthrough with a portable microscope mounted on the heads of rats (Helmchen et al., 2001), but this prototype is too heavy for mice. The wide availability of disease models makes mice the animal of choice for biomedical applications, therefore further engineering effort focused on miniaturizing all components. A mouse prototype weighing 3.9 grams and hanging slightly over the mouse’s shoulders achieved a lateral resolution of 1.2 microns (Flusberg et al., 2005; see movie). Further micromachining and electrical engineering led to a second-generation microscope that has a smaller footprint, weighs 2.5 grams, as well as optical improvements (Piyawattanametha et al., 2006). This instrument took a month to assemble once all parts were made.
The most recent device weighs 1.8 grams and does not extend past the mouse’s head. It features three micro-lenses with a lateral resolution of 0.9 micrometers, enough to see dendrites though not dendritic spines. This device enables imaging while the mouse carrying it walks about the cage. A calcium-sensitive indicator images spikes as the mouse moves. This is the first device that combines the concepts of chronic imaging and imaging of freely moving mammals. Motion artifacts so far have proven minimal.
In principle, this microscope can be used to introduce light to excite neurons in ChR2-expressing and similar transgenic mice, provided separate spectra can be used to excite the channels and the fluorophore. Quantum dots would become attractive sources of fluorescence once their targeted delivery could be worked out. For more on technologies to illuminate genetically targeted brain circuits, see Deisseroth et al., 2006. This freely downloadable review features a picture of the portable microscope.
A third imaging method discussed here has similarly completed proof of principle and is established enough to be applied to questions of AD research. It is currently undergoing technical refinement to make it cheaper, smaller, and more readily accessible to investigators either at their own respective institutions or through a service core. Array tomography is a structural imaging technique that combines advanced features of optical fluorescence and electron microscopy to render exquisitely detailed, high-resolution, 3D views of synapses in blocks of cortical tissue.
With array tomography, the experimenter fixes a specimen—e.g., a piece of fresh human cortex excised during aneurysm surgery, or a piece of APP/PS-transgenic mouse brain—in acrylic resin and cuts it with a specially devised technique into bands of hundreds or thousands of consecutive serial sections 50 to 200 nm thick. Two innovations in these initial steps lie in automating the cutting of the series without losing any, and gathering them on coated glass microscope slides where they are amenable to repeated future manipulations and are generally much more robust than serial sections cut for conventional electron microscopy. Another innovation lies in optimizing the resin toward post-embedding staining with fluorescent antibodies, not immunogold as used in conventional TEM.
Next, hundreds of consecutive sections on a slide are labeled with antibodies, and then a fluorescent microscope is fully automated to take individual pictures of the same view section after section. Software is available to align the images into data stacks that can either be analyzed quantitatively or visualized in 3D.
For example, array tomography resolved, and then counted, 280,000 individually visible synapses stained with synapsin-1 in the synaptic neuropil of a sample of mouse whisker barrel cortex. This tissue block represented 1/300th of the entire mouse whisker barrel structure, and the data was collected in 1 hour of fully automated imaging. The density of synapses rendered in this way corresponds to estimates made previously with unbiased stereologic counts. In an example relevant to AD, array tomography imaged within 2 days an amyloid plaque with a halo of missing synapses in a Tg2576 mouse. A previously generated multiphoton image of the same plaque had taken months to generate computationally (Skoch et al., 2005). Array tomography could be combined with multiphoton microscopy to assess in more detail how plaques affect nearby synaptic architecture as a function of time.
Array tomography offers two further advantages. First, antibody stripping and repeated application of different labeling antibodies work robustly, having been tested in nine cycles of antibody staining, imaging, and elution on the same band of sections (Micheva and Smith, 2007). This allows for multiplexing and detailed immunohistochemical characterization of a given cortical area without degrading the antigens in the sections. Second, after fluorescent imaging, the same sections can be imaged with other modalities, e.g., backscattered electron detector scanning electron microscopy (BSE-SEM). Voxel registration provides images of a given synapse with fluorescent microcopy side-by-side with an SEM image that approaches high-magnification transmission electron microscopy (TEM) in its resolution.
These two features open up the possibility of classifying individual synapses, i.e., by their neurotransmitter or postsynaptic receptor subunits. Used in transgenic mouse models, array tomography can classify which synapses are most susceptible to toxicity by Aβ. It can also tackle more broadly the problem of differential sensitivity of neuronal populations to AD pathology in molecular ways. A given tissue block can be sequentially probed for the presence of GluR2, of GABA, ChAT, specific forms of hyperphosphorylated tau, etc., and rendered in a joint image.
In contrast to array tomography, optical sectioning with confocal imaging is widely available. It can approach similar questions to a rough approximation. However, poor penetration of antibodies into deep tissue, as well as incontrovertible physical limits imposed by the point spread function, both curtail its efficiency of imaging along the Z axis, i.e., into the depth of tissue. Physical sectioning, post-embedding staining, and automated microscopy and image processing open up a new level of analysis in tissue imaging. —Gabrielle Strobel.
See also Part 1, Part 2, Part 3, and Part 5.