Manganese ion (Mn2+) is paramagnetic, allowing its detection in spin-lattice (T1) -weighted magnetic resonance imaging (MRI) images. This property makes Mn2+ an extremely important potential tool. Mn2+ enters neurons via calcium (Ca2+) channels and, once inside the cells, is sequestered in the endoplasmic reticulum, subsequently transported along microtubules via fast axonal transport and released at the synaptic cleft. We developed the technique of Manganese Enhanced MRI (MEMRI) neuronal tract tracing to allow the in vivo, trans-synaptic visualization of traced neuronal pathways within the CNS. These tracings can be acquired without harming the animal, and the Mn2+ ion washes out of the tissue after several days allowing longitudinal studies on the same animal. We have successfully produced MEMRI tract tracings in the olfactory system, visual system, striatum, and amygdala in mice and in the frontal cortex of rhesus macaques.
Axonal transport deficits have been observed in flies and cultured rodent neurons exposed to excess amyloid precursor protein (APP) or amyloid-beta (Ab), but neither the molecular basis of the transport deficit nor the temporal relationship of the transport deficit and the acquisition of Alzheimer's Disease (AD) are known. Additionally, Presenilin 1 is an important gene in the acquisition of Alzheimer's Disease as it is thought to be involved in the development of early Alzheimer's Disease. Mice expressing mutant presenilin (PS1M146V mice) accumulate Ab and develop plaques at an accelerated rate.
Because Mn2+ is transported along microtubules via fast axonal transport, it is possible to utilize MRI to dynamically measure the rates of Mn2+ transport, reflective of fast axonal transport rates within the same animal before and during disease progression.
Because we will be examining structures connected via fiber tracts, measurement of the changes in signal intensity due to Mn2+ accumulation are reflective of the rate that the Mn2+ is being transported. Our group, Nikos Logothetis' group, and Annemie Van der Linden's group have all reported the rate of Mn2+ transport at ~ 2mm/hour. Assuming no partial volume or spin-spin relaxation (T2) effects, the local concentration of Mn2+ will be proportional to the shortening of the spin-lattice (T1) relaxation rate in a specified ROI. Although the spin echo intensity has an exponential dependence on 1/T1, we are assuming that the change in concentration of the Mn2+ ion covers a small enough portion of this spread such that it will appear linear. Therefore, any changes in signal intensity that we observe should be reflective of axonal transport of Mn2+ from the injected structure to the directly connected structure.
Our hypotheses are: 1) Normal aging mice will exhibit declines in axonal transport rates throughout the brain as aging ensues; 2) The presence of excess APP and mutant presenilin 1 causes a reduction in axonal transport prior to the formation of neuritic plaques; and 3) Impairment in the sequestering of Mn2+ into the endoplasmic reticulum results in the observed early decline in axonal transport rates.
The overall goals of this application are: 1) to determine if a decline in in vivo axonal transport rates in neurons residing in the central nervous system (CNS) occurs in normal aging mice; 2) to determine if transgenic mice overexpressing the familial AD (FAD) mutant of amyloid precursor protein (APP) as well as mutant presenilin 1 (PS1M146V mice) exhibit a significant difference in in vivo axonal transport rates from the observed decline in age-matched controls during the progression of plaque formation; 3) to determine the mechanism of the decline in axonal transport by identifying which steps in Mn2+ uptake or transport are affected.
The basic format of the experimental procedure is as follows: 1) Administration of the Mn2+ to the targeted areas of the CNS; 2) MRI imaging will occur 1 hour after Mn2+ administration of the targeted area and will last for one hour and twenty minutes, totaling approximately 40 images per dynamic data set. Each image takes approximately 2 minutes to acquire. An n = 5 control mice and n = 5 for the AD mice will be collected for each age point. All mice will be age and sex matched. The zero time point for imaging will be one hour after nasal lavage; 3) Analysis of the MRI data to ascertain the rate of transport of the Mn2+ ion.
MRI Imaging: T1-weighted, spin-echo 2D data sets will be acquired of the mouse brain using a horizontal bore 9.4T Bruker Avance imaging spectrometer with a micro-imaging gradient insert and a 35 mm volume coil (Bruker Instruments,Billerica, MA). The imaging parameters are as follows: Multi-Slice/Multi Echo 2D imaging protocol, matrix dimensions = 128x128; FOV = 1 cm x 1; slice thickness = 1 mm, resulting in an in-plane spatial resolution of 78 mm. The repetition time (TR) = 504.1ms; echo time (TE) = 8.2 ms and NA = 2. The total imaging time per image is 2 minutes. A total of 40 image sets will be collected. The short TR ensures a heavily T1-weighted image that will provide positive signal enhancement in regions with an accumulation of the paramagnetic Mn2+. Because axonal transport is a temperature dependent process, it is very important to monitor and maintain the body temperature of the mouse. The body temperature of the mouse will be monitored and maintained at 36.90 C utilizing a heated, circulating water blanket.