The minimal dose of MnCl2 that produced reversible toxicity and MR signal
Previous tracing with MEMRI in the visual system has typically used volumes of 1-3 μl and MnCl2
concentrations up to 1.0 M for injection into either the anterior chamber of the mouse eye (Lindsey et al., 2007
) or the posterior chamber of the rat eye(Thuen et al., 2005
). We noted that the eyes of animals so injected turned gray, and physical exam suggested that they could no longer see. We thus set out to measure visual light response as a means to determine functional effects of the injection of MnCl2
on the retinal neurons and the visual system.
Light is detected in the retina by photoreceptors, which transmit signals to retinal ganglion cells that then send electrical potentials along the optic tract to the visual cortex. The electrical potential evoked by light can be measured over the visual cortex of the brain with externally placed electrodes and provides a measure of whether the visual pathway is intact. Thus, visual evoked potentials (VEP) measured before and after MnCl2 injection into the eye can reveal the effect of Mn2+ on electrical activity in response to light in the visual pathway ().
Mn2+ injection into the eye affects electrical activity in response to light
Before injection, the VEP of C57 (wild type) mice was robust (). Four hours after injection of 0.5 μl of 200 mM MnCl2 into the right eye, no response to light was detected even with both eyes open. Even at 24 hr after injection, only a small late potential change was detected. Thus injection in one eye affects visual responses in the entire visual track, possibly due to cation-induced hyperpolarization in the injected eye that propagates to the visual cortex rendering it unresponsive to input from either eye.
This toxicity could be either due to MnCl2 or to an increase in intraocular pressure caused by the volume of injectate. We tested a volume effect by injecting a similar amount of saline. A similar volume of saline (0.5 μl) also depressed VEPs at 4 hr after injection, but complete recovery was evident at 24 hr (). Hence, at this volume and concentration of MnCl2 there was a volumetric effect.
Smaller volumes of MnCl2, 0.25 μl, still depressed the light response at 4 hr but some return of function was apparent at 24 hr (). Even a smaller volume, 0.125 μl and lower concentration of MnCl2 (50 mM) still depressed the VEP response at 4 hr but allowed full recovery at 24 hr (). Injections of 0.25 μl of saline did not cause VEP depression (). Thus, 0.25 μl has no volumetric effect, and the VEP depression with this volume of MnCl2 is due to the ionic composition and not the volume. Unfortunately, we could not detect optic track enhancement with a lower dose of Mn2+, 0.125 μl at 50 mM. (data not shown). Thus, even doses of Mn2+ lower than can be detected by our 11.7T MR system depress VEPs at 4 hr after injection into the vitreous. It may be possible to detect MR signal from a lower Mn2+ dose with a 7T field strength. Injection into the anterior chamber could also mitigate toxicity.
Injection of 0.25 μl saline has no effect on visual evoked potentials. CBA mice with rd−/− have no response to light by VEP
Since visual response recovered at 24 hr after 0.25 μl injection of 200 mM MnCl2, and since this dose produced a contrast signal detectible by MRI in the optic pathway, we opted for a 0.25 μl injection of 200 mM MnCl2 for all subsequent experiments described here.
CBA mice had no response to light in the visual system
Preinjection VEPs detected no response to light in the visual system in 4-6 week old CBA mice positive for the rd mutation by PCR (). Similar lack of response was measured in older CBA (7-14 months old). Thus, these mice are functionally blind with no detectible electrical signals in the visual pathway by VEP.
Time-lapse MR imaging of Mn2+ transport in the optic nerve revealed no obvious difference between blind and sighted mice
To image Mn2+ during transport into the optic nerve immediately after injection into the eye, we developed a pulse sequence to capture a 32-slice slab image every 6 minutes, beginning 30 min and continuing for 2 hr to conclude at 2.5 hr after injection (). In both C57 sighted and CBA blind mice, either young (4-6 weeks) or old (10 month), Mn2+-induced signal enhancement appeared along the optic nerve over the first 2.5 hr with bright delineation of the optic tract at 24 hr.
Time-lapse MRI of optic nerve enhancement after Mn2+ injection into the right eye of sighted and blind mice
At 30 minutes after injection MR images displayed hyper-intense signal in the vitreous of the eye that gradually decreased with time as intensity increased along the optic nerve (). At 30 min, signal was present in the optic nerve emerging from the eye. Over the next 2 hr, signal along the optic tract gradually increased, with no defined wave front. The rate of increase in Mn2+
-induced signal enhancement along the optic nerve is dependent on uptake into the ganglion cells in the retina, accumulation inside the cell, and transport along their axons. Signal enhancement was detectible at the chiasm, ~1.2 cm from the eye, at 2.5 hr suggesting a minimal transport rate of 5 mm/hr consistent with fast anterograde transport of organelles in the optic system (Brady et al., 1982
; Lavail et al., 2005
; Martin et al., 1999
; Satpute-Krishnan et al., 2003
; Shah and Cleveland, 2002
The rate of appearance of Mn2+
signal along the optic nerve appeared similar in sighted and blind mice at all ages studied. This similarity demonstrates that uptake and transport are independent of electrical activity in the eye and along the optic nerve. Thus while Mn2+
can enter through voltage-dependent calcium channels(Merritt et al., 1989
), it may also enter neurons through other ion channels, such as divalent metal transporters(Thompson et al., 2007
). Once inside the cell, Mn2+
must complex with other molecules or enter subcellular compartment(s) for fast transport. Different compartments are known to travel at different rates in the axon(Brady et al., 1982
; Grafstein and Forman, 1980
; Satpute-Krishnan et al., 2006
; Thompson et al., 2007
; Vale et al., 1985
). For example mitochondria travel slowly (0.05-0.3 μm/sec)(Satpute-Krishnan et al., 2003
) while transport vesicles in neurons may travel as fast as 5 μm/sec (Vale et al., 1985
may enter more than one of these compartments, and thus travel at various rates in the same axon.
Fewer axons are found in the optic nerve of injected eyes
Optic nerves from CBA and C57 mice fixed by perfusion several months after eye injections were examined by histology () and electron microscopy (). By both whole mounts and in histology, the diameter of the optic nerve on the Mn2+ injected side was 6% less than the un-injected side (). This was true in either the blind CBA or the sighted C57 mouse. Histologic examination at higher magnification () suggested that in addition to decreased optic nerve diameter, there was a loss of axons and a replacement by glial or other repair tissue. Counts of axon numbers at higher magnification of histologic sections revealed 391 +/− 17 axons per 4.5 ×103 μm2 in the optic track of the un-injected (left) side of a C57 sighted mouse (), and similar numbers in un-injected CBA mice. After injection, both CBA and C57 mice displayed fewer axons per cross sectional area of the optic nerve, with only 291 +/−4.6 axons per 4.5 ×103 μm2 in the optic nerve of C57 mice and 281 +/− 4.7 axons in CBA. Thus injection into the eye results in ~25% decrease in the number of axons per unit area in either blind or sighted mice. No inflammation or infection was observed in any of the histologic sections. Saline injected axons were not examined, as similar volumes produced no VEP defect and thus no axonopathy is expected.
Long-term effects of Mn2+ injection by histology
Ultrastructure of axons in the optic nerve from Mn2+ injected eyes
By electron microscopy () axons in the optic nerve of injected eyes from either C57-sighted or CBA-blind mice were comparable, with similar myelination and equivalent presence of neurofilaments, microtubules and mitochondria. CBA axons appeared normal in all morphological respects despite the lack of electrical stimulation from photoreceptors. Thus ultrastructural analysis of surviving axons revealed no evidence of on-going toxic effects at this time point after injection.
Transport is slowed but not abolished in KLC1−/− mice
To test whether this form of kinesin was involved in Mn2+
transport, we used KLC1 knockout mice that lack this type of light chain(Rahman et al., 1999
; Vale et al., 1985
). These mice have alterations in kinesin behavior. At 24 hr after injection into the right eye, enhancement of the optic tract in KLC1 knockouts was similar to littermates. However at earlier time points Mn2+
enhancement developed more slowly (). At 2.5 hr after injection, the optic track remained difficult to identify in KLC1 −/− mice, even though in all other mice assessed enhancement was obvious by this time point. Because accumulation of signal is gradual, this difference might not have been detected had we only captured 24 hr images.
Time-lapse MRI of optic nerve enhancement after Mn2+ injection into the right eye of KLC+/+ (wildtype littermate) and KLC −/− mice
enters cells and associates either with protein complexes or membranes, it may associate with a subset of cargo complexes that depend on KLC1 for their transport(Bowman et al., 2000
; Kamal et al., 2000
; Verhey et al., 2001
). The lack of KLC1 in mutants would thus produce a decrease in the number and/or speed of Mn2+
-containing vesicles being transported and thus a delay in the early wave of Mn2+
transport towards the chiasm, as observed here in the time-lapse sequence. By 24 hr this slower accumulation was sufficient to produce signal along the optic tract similar to C57/b6 and to littermates with KLC1+/+ genotype. Hence similarity at later time points to wildtype may be explained by the transport of Mn2+
in KLC1−/− being mediated by motors other than conventional kinesin with the KLC1 light chain.
ROI analysis of the intensity changes in the optic track during the first 2.5 hr after injection demonstrate that in KLC+/+ intensity increased at 1 cm from the eye by 150% while in KLC−/− no increase is detected (). Unpaired students t-test demonstrated that this difference in intensity has a probability of 0.0001 of occurring by chance.
Repair is depressed in KLC1 knockout mice
VEP analysis of KLC1 knockouts revealed a defect in restoration of the normal light response at 24 hr after Mn2+ injection (). In normal littermates, VEPs at 24 hr were analogous to pre-injection depolarizations after right eye injection, with only a slight delay in response and minor alterations in voltage restitution after depolarization (). In KLC knockouts, at 24 hr after Mn2+ injection into the right eye, voltage change in response to light was both delayed and depressed (). Thus, these KLC1 −/− animals do not repair the visual system as completely as the other genotypes surveyed here, which could also be a consequence of decreased transport.
Trans-synaptic tracing by Mn2+ requires neuronal activity
Previous studies have demonstrated that Mn2+
signal crosses synapses and thus traces circuits beyond those of single neuronal projections(Murayama et al., 2006
; Pautler et al., 2003
; Saleem et al., 2002
). To determine whether this trans-synaptic transmission was dependent on neuronal activity, studies have relied on experiential stimulation of the particular pathway involved. However, to apply Mn2+
tract tracing to unknown pathways, it will be important to know definitively whether trans-synaptic transmission requires neuronal activity. The blind mice used in this study provide a more rigorous test for this analysis.
Three dimensional whole brain images of mouse brains 24 hr after Mn2+ injection into the eye resulted in Mn2+-induced enhancement in the midbrain of KLC1+/+ wildtype, and KLC−/− mice, despite the initial slower transport in the knockouts (). However, in blind mice lacking neuronal activity in the visual system, little Mn2+ signal was detected in either the lateral geniculate nucleus or the superior colliculus in the midbrain. This is despite robust signal along the optic track and an equivalent number and caliber of axons as determined by our histological examination reported earlier in this paper (Fig. -).
Mn2+ enhancement is greater in the midbrain at 24 hr post eye injection in C57/b6, KLC −/− knockouts, than in blind CBA mice
ROI analysis of the intensity in the eye, optic track and midbrain in the three types of mice examined here (wildtype, KLC−/− and CBA) at 24 hr after eye injection () demonstrated (1) similar intensity in all three mice in the eye, (2) slight (20%) decrease intensity in the optic tract in KLC−/− and CBA compared to wildtype, (3) significantly less intensity (80-90% less) in midbrain trans-synaptic sites in blind (CBA) compared to wildtype and KLC−/− mice.
Thus, while Mn2+ uptake and transport within the neuron occur independent of electrical activity, Mn2+ is not transmitted efficiently across synapses in the absence of electrical activity in this system.