The methods are a combination of functional and structural micro MRI (in vivo mice imaging) with in vitro neuronal-(axon and dendrites) calcium imaging and basic electrophysiology of the hippocampus.
Our main hypothesis is that cognitive deficits in ageing and some neurobehavioral entities are related to altered central nervous system calcium homeostasis, in specific subregions of the hippocampal formation. Since the MRI-techniques (Steady-State Cerebral Blood Volumes) that we have developed in mice can be safely applied to humans, we have started a series of collaboration imaging humans with a variety of neurocognitive deficits.
Almost all in-vivo techniques that measure brain metabolism are based on Fickís principle. This principle describes a relationship between oxidative metabolism and hemodynamic variables ó cerebral blood flow (CBF), cerebral blood volume (CBV) and deoxyhemoglobin (dHb) ó to assess metabolism in the living brain. These techniques include: Near infrared spectroscopy, contrast enhanced computerized tomography to evaluate cerebral blood volume (CBV), PET and single-photon emission tomography measurements of cerebral blood flow (CBF), and magnetic resonance imaging (MRI) measurements of CBF, CBV, and deoxyhemoglobin.
We have focused our attention on MRI-CBV because it can visualize small structures such as the hippocampal subregions with better resolution
n than other techniques such as PET. The basic idea is that a change in MRI signal caused by a shift in the intravascular concentration of a contrast agent is proportional to local blood volume. All data is presented as normalized ΔR2 values=rCBV, relative CBV. ΔR2 is the change in relaxation rate (1/T2), mainly in the microvasculature, induced by a contrast agent. All MRI experiments are performed with a Brucker AVANCE 400WB spectrometer, outfitted with an 89 mm bore 9.4 tesla vertical Brucker magnet.
Using MRI-CBV we identified the hippocampal areas that show abnormal metabolism at the earliest time point in AD and DS mouse models. Then, we performed two types of electrophysiological measurements: a) hippocampus (subiculum and CA3 pyramidal neurons) field and intracellular recordings to interrogate the circuit properties of these areas and their synaptic plasticity in J20 and Ts65Dn mice. This set-up is optimal to study animals of any age. b) patch-clamp, coupled with a high performance EM-CCD camera to study basic electrophysiological properties of individual subicular pyramidal neurons and their calcium signals in three compartments: soma, dendrites and axons. Since fMRI data are indirect measurements, we proposed to confirm imaging findings with independent measures of neuronal dysfunction, such as electrophysiology and calcium signaling.
Figure 1. Calcium transient at a dentate gyrus granule cell mossy fiber bouton. Shown are DG cells labeled with OG-1AM, before and after a 50 Hz train of 8 action potentials.
Figure 2. Mouse hippocampal subregional CBV maps. Axial T2 weighted image at the level of the midbody of the hippocampus. Shown are: entorhinal cortex, red; subiculum, blue; CA1, green; CA3, yellow; dentate gyrus, light blue.
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Sergio Angulo, M.D., Postdoctoral Fellow
Daniel Vela, M.D., Postdoctoral Fellow
Reviewer, Alzheimer Association Grants
Permanent Faculty Latin-American IBRO course on Cellular and Molecular Neurobiology