Role of neuronal activity and kinesin on tract tracing by manganese-enhanced MRI (MEMRI)

Abstract

MEMRI offers the exciting possibility of tracing neuronal circuits in living animals by MRI. Here we use the power of mouse genetics and the simplicity of the visual system to test rigorously the parameters affecting Mn2+ uptake, transport and trans-synaptic tracing. By measuring electrical response to light before and after injection of Mn2+ into the eye, we determine the dose of Mn2+ with the least toxicity that can still be imaged by MR at 11.7 T. Using mice with genetic retinal blindness, we discover that electrical activity is not necessary for uptake and transport of Mn2+ in the optic nerve but is required for trans-synaptic transmission of this tracer to distal neurons in this pathway. Finally, using a kinesin light chain 1 knockout mouse, we find that conventional kinesin is a participant but not essential to neuronal transport of Mn2+ in the optic tract. This work provides a molecular and physiological framework for interpreting data acquired by MEMRI of circuitry in the brain.

Figure 1. Mn²⁺ injection into the eye affects electrical activity in response to light

Shown are graphs of averaged VEP measurements before, 4 hr, and 24 hr after injection of various volumes of Mn²⁺ or saline solutions into the right eye of sighted C57/b6 mice (A-D). In Fig.1A, VEP was measured with both eyes open. In B-D, VEPs were measured from the injected right eye (red), the un-injected left eye (yellow), both eyes open (dark blue) or both eyes closed (light blue). Full recovery occurred at 24 hr at lower doses.

Figure 2. Injection of 0.25 μl saline has no effect on visual evoked potentials. CBA mice with rd−/− have no response to light by VEP

(A) Injection of 0.25 μl saline had no effect on VEP of C57 wildtype mice at 4 hr (color code is the same as in Fig. 1). (B) Preinjecton VEP measurements of three different CBA mice with both eyes open demonstrated that they were functionally blind (CBA: red, yellow, and dark blue) with no light response as compared to a C57 mouse tested on the same day (C57: light blue).

Figure 3. Time-lapse MRI of optic nerve enhancement after Mn²⁺ injection into the right eye of sighted and blind mice

Shown are representative images of four of the mice used in this study, two sighted C57 and two blind CBA. In each pair, the top sequence is from a young (6-8wk old) mouse and the lower from an older (7 mo old) mouse. Images were captured at every 6 min. Shown are images selected from these time-lapse sequences, beginning with the first, captured at 30 min post-injection, and then at 1 hr, 1.5 hr and 2.5 hr post injection. At 24 hr after injection a similar slab image bracketing the optic tract was captured. Similar images were obtained for all 5 CBA young, 5 CBA old and 7 C57 mice of any age.

Figure 4. Long-term effects of Mn²⁺ injection by histology

Optic nerve from an uninjected C57 mouse at low magnification (A) and high magnification (B), and optic nerves from injected eye of C57 (C and E) and of CBA blind mouse (D and F).

Figure 5. Ultrastructure of axons in the optic nerve from Mn²⁺ injected eyes

Representative examples of axons imaged by TEM within the right optic nerve of sighted C57 mouse (A) and blind CBA mouse (B) each injected with Mn²⁺ in the right eye are shown. Note the dark myelin sheath encircling the axons, the dots within the axons representing cytoskeletal elements in cross section, and the presence of cytoplasmic organelles within the axons (arrowheads).

Figure 6. Time-lapse MRI of optic nerve enhancement after Mn²⁺ injection into the right eye of KLC+/+ (wildtype littermate) and KLC −/− mice

A) Representative images from two time-lapse sequences, KLC+/+ (top) and KLC −/− mice, captured at 30 min, 1, 1.5 and 2.5 hr after injection of 0.25 μl of 200 mM Mn²⁺ into the right eye. Note that by 2.5 hr after injection in the littermate, Mn²⁺ signal is present along the optic nerve possibly reaching the chiasm. In contrast in the KLC−/− mouse, Mn²⁺ enhancement is only detected in a short segment at the beginning of the optic nerve. B) At 24 hr Mn²⁺ signal has progressed along the optic track in both genotypes, demonstrating that the optic tract is intact. C) ROI intensity measurement (position indicated by circles in left panel) demonstrate that over the first 160 min after Mn²⁺ injection into the vitreous, intensity (the ratio of optic track vs. cheek muscle) increased in the optic tract by 150% in the wildtype KCL++ while no intensity increase was detected in the KLC−/− mouse. Error bars represent the sum of the standard deviations of the average voxel intensities in optic tract vs cheek muscle ROIs. For KLC+/+ these were 10% and 6 % respectively, and for KLC−/− 7% and 6%.

Figure 7. VEP of KLC mice

Shown are examples of VEPs from two mice, one KLC +/+ littermate (A) and one KLC −/− knockout (B) before (blue) and at 24 hr (magenta) after Mn²⁺ injection into the right eye. Note the delayed response to light after Mn²⁺ injection in both types of mouse, and the significant decrease in signal intensity in the knockout at 24 hr.

Figure 8. Mn²⁺ enhancement is greater in the midbrain at 24 hr post eye injection in C57/b6, KLC −/− knockouts, than in blind CBA mice

A) Coronal sections through the midbrain taken from 3D MR images captured at 24 hr post Mn²⁺ injection into the right eye are shown for mouse genotypes as indicated. Sections through the lateral geniculate (left panels) or the superior colliculus (right panels) demonstrate Mn²⁺ induced increased intensity in C57 and KLC1−/− but no obvious increase in the blind CBA mouse. B) ROI analysis of intensity in the eye, optic track, and midbrain (lateral geniculate nucleus (LGN); and superior colliculus (sup coll). Wildtype (KLC+/+) is shown in blue, KLC −/− in red, and rd−/− (blind) mice in yellow. Intensity values represent the ratio of average voxel intensity in optic tract vs. standard tube, Error bars represent the sum of the standard deviations of voxel intensities of the ROIs and the standard tube.