Manganese‐enhanced MRI optic nerve tracking: effect of intravitreal manganese dose on retinal toxicity

The aim of this study was to provide data on the dose dependence of manganese‐enhanced MRI (MEMRI) in the visual pathway of experimental rats and to study the toxicity of MnCl₂ to the retina. Sprague–Dawley rats were intravitreally injected with 2 μL of 0, 10, 25, 50, 75, 100, 150 and 300 mm MnCl₂, respectively_. The contrast‐to‐noise ratio (CNR) of MEMRI for optic nerve enhancement was measured at different concentrations of MnCl₂. Simultaneously, the toxicity of manganese was evaluated by counting retinal ganglion cells and by retinal histological examination using light microscopy and transmission electron microscopy. The CNR increased with increasing concentration of MnCl₂ up to 75 mm . Retinal ganglion cell densities were reduced significantly when the concentration of MnCl₂ in the intravitreal injection was equal to or greater than 75 mm . Increasing numbers of ribosomes in retinal ganglion cells were first detected at 25 mm of MnCl₂. The retinal toxicity of MnCl₂ at higher concentration also included mitochondrial pathology and cell disruption of retinal ganglion cells, as well as abnormalities of photoreceptor and retinal pigment epithelium cells. It can be concluded that intravitreal injection of MnCl₂ induces retinal cell damage that appears to start from 25 mm . The concentration of MnCl₂ should not exceed 25 mm through intravitreal injection for visual pathway MEMRI in the rat. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of paramagnetic manganese cations on (1)H MRS of the brain

Manganese cations (Mn(2+)) can be used as an intracellular contrast agent for structural, functional and neural pathway imaging applications. However, at high concentrations, Mn(2+) is neurotoxic and may influence the concentration of (1)H MR-detectable metabolites. Furthermore, the paramagnetic Mn(2+) cations may also influence the relaxation of the metabolites under investigation. Consequently, the purpose of this study was to investigate the effect of paramagnetic Mn(2+) cations on (1)H-MR spectra of the brain using in vivo and phantom models at 4.7 T. To investigate the direct paramagnetic effects of Mn(2+) cations on the relaxation of N-acetylaspartate (NAA), creatine and choline, T(1) relaxation times of metabolite solutions, with and without 5% albumin, and containing different Mn(2+) concentrations were determined. Relaxivity values with/without 5% albumin for NAA (4.8/28.1 s(-1) mM(-1)), creatine (2.8/2.8 s(-1) mM(-1)) and choline (1.8/1.1 s(-1) mM(-1)) showed NAA to be the most sensitive metabolite to the relaxation effects of the cations. Using an in vivo optic tract tracing imaging model, we obtained two adjacent regions of interest in the superior colliculi with different water T(1) values (Mn(2+)-enhanced = 1.01 s; unenhanced = 1.14 s) 24 h after intravitreal injection of 3 microL 50 mM MnCl(2). Using phantom and in vivo water relaxation time data, we estimated the in vivo Mn(2+) concentration to be 2-8 microM. The phantom data suggest that limited metabolite relaxation effects would be expected at this concentration. Consequently, this study indicates that, in this model, the presence of Mn(2+) cations does not significantly affect (1)H-MR spectra despite possible toxic and paramagnetic effects.     

Three-dimensional MRI of cerebral projections in rat brain in vivo after intracortical injection of MnCl2

In this study we investigated the potential of in vivo MRI detection of axonal Mn2+ transport for tracing neuronal projections originating in the sensorimotor cortex in healthy and lesioned rat brains. Special attention was given to the potential of visualizing neuronal sprouting of central nervous system across the midline. After injecting unchelated MnCl2 into the forelimb area of sensorimotor cortex of 18 healthy and 10 lesioned rats corticofugal projections could be traced through the internal capsule to the cerebral peduncle and the pyramidal decussation. Although the neuronal tract was visible as early as 6 h after MnCl2 injection, best contrast was achieved after 24-48 h. Beside the cortico-spinal tract, the cortico-thalamic fibres were also visualized by anterograde Mn2+ transport. Cortico-striatal fibres were partially masked by the very high signal near the MnCl2 injection site but could be discerned as well. Slight, diffuse signal enhancement of cortical tissue contralateral to the MnCl2 injection site in healthy rat brains suggests interhemispheric connections or passive diffusion of Mn2+. However, enhanced fibre tract contrast connecting both hemispheres was visible 16 weeks after onset of focal photothrombotic cortical injury. In conclusion our study has shown that we were able to visualize reproducibly the main descending corticofugal projections and interhemispheric connections by non-invasive MRI after localized injection of MnCl2. The appearance of interhemispheric Mn2+-enhanced fibres after photothrombotic focal injury indicates that the method may bear potential to follow non-invasively gross plastic changes of connectivity in the brain after injury.     

Manganese threonine chelate—a new enteric contrast agent for MRI: a pilot study on rats

Enteric contrast agents are important in gastrointestinal MRI. However, no currently available agent is well established as the standard of care. In this study, in vitro relaxivities of manganese threonine chelate (Mn‐Thr), a common nutritional food supplement, were measured at 1.5 T and 3 T with further investigation of its efficacy and safety in vivo as an enteric contrast agent. According to the calculated relaxivities, T₁W and T₂W TSE sequences of Mn‐Thr solutions at different concentrations were acquired, and the optimal concentration for dark lumen imaging on both T₁W and T₂W images was determined in vitro. To validate the optimal concentration in vivo, eight Sprague‐Dawley rats were randomly divided into two groups. Each group received rectal injection of either 2.00 g/L (about 3.80 mM) Mn‐Thr or saline as an enteric contrast agent and underwent MRI. After a time interval of one week, the same procedures were repeated with the alternative contrast agent. Animals were sacrificed after the second MRI. Tissue manganese quantification and histopathological examination were obtained. Qualitative MR image quality assessments were performed and compared between Mn‐Thr and saline. Measured T₁ and T₂ relaxivities of Mn‐Thr were significantly higher than those of MnCl₂ in vitro (p < 0.05). At the concentration of 2.00 g/L (about 3.80 mM), Mn‐Thr produced a dark lumen on T₁W and T₂W images both in vitro and in vivo. Compared with saline, Mn‐Thr showed significantly more homogenous luminal signal and increased bowel wall conspicuity in image quality assessments. Tissue manganese concentrations were not significantly different between two groups. Histopathological examinations were normal in both groups. Our data suggest that Mn‐Thr possesses favorable paramagnetic properties and can create a homogenous dark lumen on T₁W and T₂W images without obvious side effects in healthy rats. As a commercially available nutritional food supplement, Mn‐Thr appears to be a promising enteric contrast agent for MRI.     

In vivo manganese-enhanced magnetic resonance imaging reveals connections and functional properties of the songbird vocal control system

Injection of manganese (Mn(2+)), a paramagnetic tract tracing agent and calcium analogue, into the high vocal center of starlings labeled within a few hours the nucleus robustus archistriatalis and area X as observed by in vivo magnetic resonance imaging. Structures highlighted by Mn(2+) accumulation assumed the expected tri-dimensional shape of the nucleus robustus archistriatalis and area X as identified by classical histological or neurochemical methods. The volume of these nuclei could be accurately calculated by segmentation of the areas highlighted by Mn(2+). Besides confirming previously established volumetric sex differences, Mn(2+) uptake into these nuclei revealed new functional sex differences affecting Mn(2+) transport. A faster transport was observed in males than in females and different relative amounts of Mn(2+) were transported to nucleus robustus archistriatalis and area X in males as compared to females.This new in vivo approach, allowing repeated measures, opens new vistas to study the remarkable seasonal plasticity in size and activity of song-control nuclei and correlate neuronal activity with behavior. It also provides new insights on in vivo axonal transport and neuronal activity in song-control nuclei of oscines.     

Manganese enhancement in non-CNS organs

Manganese-enhanced magnetic resonance imaging (MEMRI) is a novel imaging technique capable of monitoring calcium influx, in vivo. Manganese (Mn2+) ions, similar to calcium ions (Ca2+), are taken up by activated cells where their paramagnetic properties afford signal enhancement in T(1)-weighted MRI methodologies. In this study we have assessed Mn2+ distribution in mice using magnetization-prepared rapid gradient echo (MP-RAGE) based MRI, by measuring changes in T(1)-effective relaxation times (T(1)-eff), effective R(1)-relaxation rates (R(1)-eff) and signal intensity (SI) profiles over time. The manganese concentration in the tissue was also determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). Our results show a strong positive correlation between infused dose of MnCl2 and the tissue manganese concentration. Furthermore, we demonstrate a linear relationship between R(1)-eff and tissue manganese concentration and tissue-specific Mn2+ distribution in murine tissues following dose-dependent Mn2+ administration. This data provides an optimized MnCl2 dose regimen for an MP-RAGE based sequence protocol for specific target organs and presents a potential 3D MRI technique for in vivo imaging of Ca2+ entry during Ca2+-dependent processes in a wide range of tissues.     

Longitudinal manganese-enhanced magnetic resonance imaging of neural projections and activity

Manganese-enhanced magnetic resonance imaging (MEMRI) holds exceptional promise for preclinical studies of brain-wide physiology in awake-behaving animals. The objectives of this review are to update the current information regarding MEMRI and to inform new investigators as to its potential. Mn(II) is a powerful contrast agent for two main reasons: (1) high signal intensity at low doses; and (2) biological interactions, such as projection tracing and neural activity mapping via entry into electrically active neurons in the living brain. High-spin Mn(II) reduces the relaxation time of water protons: at Mn(II) concentrations typically encountered in MEMRI, robust hyperintensity is obtained without adverse effects. By selectively entering neurons through voltage-gated calcium channels, Mn(II) highlights active neurons. Safe doses may be repeated over weeks to allow for longitudinal imaging of brain-wide dynamics in the same individual across time. When delivered by stereotactic intracerebral injection, Mn(II) enters active neurons at the injection site and then travels inside axons for long distances, tracing neuronal projection anatomy. Rates of axonal transport within the brain were measured for the first time in “time-lapse” MEMRI. When delivered systemically, Mn(II) enters active neurons throughout the brain via voltage-sensitive calcium channels and clears slowly. Thus behavior can be monitored during Mn(II) uptake and hyperintense signals due to Mn(II) uptake captured retrospectively, allowing pairing of behavior with neural activity maps for the first time. Here we review critical information gained from MEMRI projection mapping about human neuropsychological disorders. We then discuss results from neural activity mapping from systemic Mn(II) imaged longitudinally that have illuminated development of the tonotopic map in the inferior colliculus as well as brain-wide responses to acute threat and how it evolves over time. MEMRI posed specific challenges for image data analysis that have recently been transcended. We predict a bright future for longitudinal MEMRI in pursuit of solutions to the brain-behavior mystery.     

The effect of intraocular injection of tetrodotoxin on fast axonal transport of [3H]proline- and [3H]fucose-labeled materials in the developing rat optic nerve

The fast axonal transport of [3H]proline-labeled proteins and [3H]fucose-labeled glycoproteins delivered to the dorsal lateral geniculate nucleus in the developing rat optic nerve was investigated during tetrodotoxin-induced monocular impulse blockade. Repeated intraocular injections of various dosages of tetrodotoxin or citrate buffer vehicle were made every two days in rats aged 5-21 days postnatal, and the accumulation of rapidly transported radioactivity in the lateral geniculate nucleus measured between three and twelve hours post-injection at each age. The effectiveness of prolonged tetrodotoxin treatment was monitored by loss of the pupillary light reflex and the level of cytochrome oxidase activity in the contralateral superior colliculus and dorsal lateral geniculate nucleus. Numbers of optic axons proximal to the chiasm and the frequency of retinal ganglion cells per unit distance from the optic disc were examined for signs of tetrodotoxin-induced degeneration of the retinofugal pathway. Tetrodotoxin-treatment reduced the amount of fucosyl glycoproteins, but not proline-labeled proteins, axonally transported to the lateral geniculate nucleus during the first three weeks of postnatal development. Other studies indicated that tetrodotoxin significantly reduced the incorporation of [3H]fucose into retinal proteins indicating that the reduction in transport was probably due to a decrease in precursor incorporation into retinal ganglion cells. Electron microscopy of ganglion cells at 21 days revealed dilated and vacuolated Golgi cisternae associated with tetrodotoxin treatment, suggesting that tetrodotoxin may alter fucose metabolism by secondarily disrupting Golgi organization. Other protein synthetic machinery in these cells, including ribosomes and rough endoplasmic reticulum, appeared normal throughout tetrodotoxin treatment. These data indicate that Na+-dependent optic impulse activity may be indirectly related to the axonal transport of glycoproteins during early postnatal development by mediating the incorporation of precursor into glycoproteins at the Golgi apparatus and their subsequent entrance into the fast transport system.     

Ciliary neurotrophic factor promotes the regrowth capacity but not the survival of intraorbitally axotomized retinal ganglion cells in adult hamsters

Ciliary neurotrophic factor has recently been shown to promote the axonal regrowth of retinal ganglion cells into a peripheral nerve graft following an intracranial transection of the optic nerve (approximately 7 mm from the optic disc). It is unclear whether the enhancement of axonal regrowth by ciliary neurotrophic factor application correlates with the enhancement of survival of retinal ganglion cells and/or the up-regulation of expression of growth-associated protein-43 messenger RNA in retinas. The present study evaluated the regenerative capacity of retinal ganglion cells following intraorbital transection of the optic nerve (approximately 1.5 mm from the optic disc) and the attachment of a peripheral nerve to the ocular stump of the optic nerve. In addition, we have determined the survival of retinal ganglion cells and the expression of growth-associated protein-43 messenger RNA in ciliary neurotrophic factor-treated retinas following optic nerve transection. The results showed that in the ciliary neurotrophic factor-treated retinas, the number of retinal ganglion cells which had regrown axons into a peripheral nerve is about four times more than the control. In the axotomized retinas, ciliary neurotrophic factor initiated sprouting of axon-like processes at 14 and 28 days post-axotomy and up-regulated the expression level of growth-associated protein-43 messenger RNA at 7, 14 and 28 days post-axotomy. However, ciliary neurotrophic factor did not prevent the death of axotomized retinal ganglion cells. We suggest that one possible mechanism for the axonal regeneration of axotomized retinal ganglion cells by ciliary neurotrophic factor could be mediated by the up-regulation of growth-associated protein-43 gene expression and not by increasing the pool of surviving retinal ganglion cells after axotomy.     

Intraocular colchicine selectively destroys immature ganglion cells in chicken retina

Low doses of colchicine (greater than 0.5 microgram) injected into the eyes of young chickens irreversibly inhibit axonal transport of material labelled with [3H]fucose. This is due to almost total destruction of the retinal ganglion cells, while other retinal cell types, including the displaced amacrine cells found in the same retinal layer, survive the treatment. The neurotoxic effect of colchicine on chick retinal ganglion cells decreases after hatching, and it is suggested that the decrease in sensitivity may be related to myelination of the retinal ganglion cell axons.     

Bifurcating axons of retinal ganglion cells terminate in the hypothalamic suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus

At least some retinal axons afferent to the hypothalamic suprachiasmatic nucleus (SCN; a circadian oscillator) bifurcate in the optic chiasm (O.E. Millhouse, Brain Res., 137 (1977) 351-355). The termination site(s) of the axonal branch that continues in the optic tract is unknown. Injection of the fluorescent tracer, True Blue, into the SCN and the fluorescent dye, Nuclear Yellow, into the lateral geniculate complex resulted in the labeling of individual retinal ganglion cells with both tracers. However, only Nuclear Yellow injections which included the intergeniculate leaflet (IGL) resulted in double-labeled ganglion cells in the retinae. These results indicate that individual retinal ganglion cells innervate both the hypothalamic SCN and the IGL of the thalamus by means of divergent axonal collaterals. Moreover, neurons of the IGL are afferent to the SCN, thereby forming a complex circuit within which photic information from the same retinal ganglion cell may influence the SCN both directly and after thalamic processing.     

Anatomical correlations of intrinsic axon repair after partial optic nerve crush in rats.

About 15% of retinal ganglion cells survive diffuse axonal injury of the optic nerve in adult rats. Following initial blindness, discrimination of visual stimuli in behavioral tests recovers within three weeks. To investigate the mechanisms promoting this functional recovery the axonal transport and the neurofilaments were studied. Intraocularly applied MiniRuby is transported until the place of crush and accumulated in enlarged axon terminals. Three weeks after lesion the anterograde transport of MiniRuby recovers distal to the place of crush. At the same point in time the retrograde transport of surviving retinal ganglion cells is restored which was visualized by horseradish peroxidase injected into the superior colliculus. The heavy neurofilament was stained immunohistochemically and analyzed statistically up to three weeks after optic nerve crush. The stained filaments in the axon fibers of retinal ganglion cells appear wavelike and/or fragmented up to day 8, but first signs of heavy neurofilament restitution in the fibers of the optic nerve are seen at day 12 after axonal injury. Because these results cannot be explained by longlasting axon regeneration, the present results provide convincing evidence for intrinsic axon repair soon after diffuse axonal injury that correlates in time with recovery of vision.     

Effects of extracellular atp on axonal transport in cultured mouse dorsal root ganglion neurons

In primary sensory neurons, extracellular ATP plays important roles in nociception and afferent neurotransmission. Here we investigated the effects of ATP on axonal transport in cultured adult mouse dorsal root ganglion neurons using video-enhanced microscopy. Continuous application (26 min) of ATP (100 microM) significantly increased axonal transport of membrane-bound organelles in anterograde and retrograde directions. All neurons tested (n=5) responded to ATP. The number of transported organelles per min began to increase within 2 min and peaked at 11-14 min after the start of ATP application, and thereafter gradually declined. The peak values in both directions were approximately 140% of the initial values before application. The P2 receptor antagonist suramin (1 mM) completely blocked the effect of ATP. Uridine 5'-triphosphate (UTP; 100 microM) produced a similar effect to ATP, with peak values at 11 min reaching 140% in both directions (n=6). ADP (100 microM; n=5), alpha,beta-methylene ATP (100 microM; n=6), or 2-methylthio ATP (100 microM; n=5) had no effect on axonal transport.Our findings indicate that extracellular ATP is able to increase axonal transport in primary sensory neurons. The equal potency of ATP and UTP with no detectable response to ADP, alpha,beta-methylene ATP, or 2-methylthio ATP suggests the possible involvement of P2Y(2) receptors. Extracellular ATP may play an important role in the modulation of axonal transport in sensory neurons.     

Manganese-enhanced magnetic resonance imaging (MEMRI) without compromise of the blood-brain barrier detects hypothalamic neuronal activity in vivo

There is growing interest in the use of manganese-enhanced MRI (MEMRI) to detect neuronal activity and architecture in animal models. The MEMRI neuronal activity studies have been generally performed either by stereotactic brain injection or by systemic administration of Mn(2+) in conjunction with the disruption of the blood-brain barrier (BBB). These approaches, however, have limited the use of MEMRI because of the procedure-related morbidity/mortality or because brain activity measured by these methods can diverge from genuine physiological responses. In this study, the hypothesis that MEMRI, performed with systemic administration of Mn(2+) without compromising the BBB integrity, is able to detect hypothalamic function associated with feeding was tested. This procedure was tested on a simple physiological condition, fasting, and with this method temporal and regional differences in Mn(2+) enhancement could be detected. It is concluded that MEMRI can be used to study hypothalamic function in the murine brain without compromising the BBB. It was also shown that region-specific Mn(2+) enhancement in the mouse brain can be modulated by fasting. More importantly, this non-invasive in vivo imaging technique is able to demonstrate differences in brain activities, previously possible only by in vitro studies.     

Axonal bifurcation of cat retinal ganglion cells as demonstrated by retrograde double labelling with fluorescent dyes

The projections of cat retinal ganglion cells were investigated using fluorescent tracers. After injection of one fluorochrome into the lateral geniculate nucleus (LGN) and the other into the superior colliculus (SC), members of all three ganglion cell classes showed double-labelled somata, indicating axonal bifurcations in the optic tract. No bifurcations were found in the optic chiasm.     

A morphometric study of the retinal ganglion cell response to optic nerve severance in the frog Rana pipiens

Summary This study examines the cell body response to axotomy of retinal ganglion cells in the frogRana pipiens. Cell soma sizes were measured in carefully matched regions of Nissl-stained wholemounted retinae after either nerve crush, nerve cut with stump separation, nerve crush with intraocular nerve growth factor (NGF) or nerve cut with NGF applied to the proximal stump. The state of axonal regeneration was also assessed in each case by anterograde transport of HRP.Following nerve crush axons crossed the lesion by 7 days, reached the chiasma by 14 days and entered the tectum around 20–30 days. The normally evenly stained ganglion cells exhibited granular Nissl staining at 7 and 10 days but very little change in soma size. From 10 to 28 days the mean retinal ganglion cell area increased by 102% and maintained this size until at least 75 days. By 102 days soma size had nearly returned to normal. A population of displaced amacrine cells retained a normal appearance and soma size throughout regeneration.Following nerve cut and stump separation the retinal ganglion cells were slightly more reactive in appearance at 7 days after crush but otherwise the soma reaction developed in a similar manner. Axon tracing revealed no extension beyond the lesion site in these animals and therefore the state of axonal growth did not affect the early soma response.NGF applied at the time of the lesion had no detectable effect on the soma reaction.Although many retinal ganglion cells re-establish contact with visual centres after axotomy in the frog, a considerable proportion die. This contrasts with both the goldfish, where all cells regenerate successfully, and various mammals, where none do so and all retinal ganglion cells die. All retinal ganglion cells in the frog undergo reactive changes similar to those of goldfish and there is no sign of the cell shrinkage seen in mammals. Therefore the cell death in frog would appear to be different from that in mammalian retina but similar to that of mammalian peripheral nerve in which chromatolysis generally preceeds death.     

Selective axonal transport in a single cholinergic axon of Aplysia--role of colchicine-resistant microtubules

Substance-specific selective axonal transport was examined in a single axon by injecting [3H]leucine and [14C]acetylcholine simultaneously into the cell body of a giant cholinergic neuron (R2) in the abdominal ganglion of Aplysia kurodai. The ganglion and attached nerves were cultured for several hours after the injection and the migration of radioactive substances along the axons of the injected neuron was examined. The substances examined were 3H labeled membrane proteins and soluble proteins synthesized in the cell body, 14C labeled bound acetylcholine formed in the cell, injected [3H]leucine and soluble [14C]acetylcholine. Membrane proteins and bound acetylcholine (plus a part of soluble acetylcholine) moved along the axon somatofugally at maximum velocities of 2.4 and 1.7 mm/h, respectively, at 25 degrees C. Soluble proteins, free leucine and most of the soluble acetylcholine did not move by fast axonal transport but diffused inside the axon of the neuron R2 at rates predicted from their expected diffusion constants in the axoplasm [Koike H. and Nagata Y. (1979) J. Physiol. 295, 397-417]. The diffusion kinetics of these substances were analysed and used for determination of true axon length, and to separate axonal transport components from diffusing components. An antimitotic drug, colchicine, selectively suppressed the axonal transport of membrane proteins but not of acetylcholine at 1-5 mM concentration, though it finally blocked the axonal transport of acetylcholine at 20 mM. When 1-5 mM colchicine was separately perfused only to the distal axon of the neuron R2, the migration of membrane proteins was stopped just proximal to the colchicine perfusion zone but acetylcholine migration was not disturbed by the drug. The moving component of acetylcholine was recovered by sucrose density centrifugation from a compartment previously reported as that of vesicular acetylcholine. As a possible mechanism of this selective axonal transport, it is proposed that there are two groups of microtubules: a colchicine-sensitive group of microtubules which may transport membrane proteins, and a colchicine-resistant group which may preferentially transport the transmitter substance acetylcholine at a slower rate.     

Calcium channel activation facilitated by nitric oxide in retinal ganglion cells

We investigated the modulation of voltage-gated Ca channels by nitric oxide (NO) in isolated salamander retinal ganglion cells with the goals of determining the type of Ca channel affected and the signaling pathway by which modulation might occur. The NO donors, S-nitroso-N-acetyl-penicillamine (SNAP, 1 mM) and S-nitroso-cysteine (1 mM) induced modest increases in the amplitude of Ca channel currents recorded with ruptured- and permeabilized-patch techniques by causing a subpopulation of the Ca channels to activate at more negative potentials. The Ca channel antagonists omega-conotoxin GVIA and nisoldipine each reduced the Ca channel current partially, but only omega-conotoxin GVIA blocked the enhancement by SNAP. The SNAP-induced increase was blocked by oxadiazolo-quinoxaline (50 microM), suggesting that the NO generated by SNAP acts via a soluble guanylyl cyclase to raise levels of cGMP. The membrane-permeant cGMP analog 8-(4-chlorophenylthio) guanosine cyclic monophosphate also enhanced Ca channel currents and 8-bromo guanosine cyclic monophosphate (1 mM) occluded enhancement by SNAP. Consistent with these results, isobutyl-methyl-xanthine (IBMX, 10 microM), which can raise cGMP levels by inhibiting phosphodiesterase activity, increased Ca channel current by the same amount as SNAP and occluded subsequent enhancement by SNAP. Neither IBMX, the cGMP analogs, nor SNAP itself, led to activation of cGMP-gated channels. N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (2 microM), a broad spectrum inhibitor of protein kinase activity, KT5823 (1 microM), a specific protein kinase G (PKG) inhibitor, and a peptide inhibitor of PKG (200 microM) blocked SNAP enhancement, as did 5'-adenylylimidophosphate (1.5 mM), a nonhydrolyzable ATP analog that prevents protein phosphorylation. A peptide inhibitor of protein kinase A (10 nM) did not block the facilitory effects of SNAP. Okadaic acid (1 microM), a phosphatase inhibitor, had no effect by itself but increased the enhancement of Ca channel current by SNAP. These results suggest that NO modulates retinal ganglion cell N-type Ca channels by facilitating their voltage-dependent activation via a mechanism involving guanylyl cyclase/PKG-dependent phosphorylation. This effect could fine-tune neural integration in ganglion cells or play a role in ganglion cell disease by modulating intracellular calcium signaling.     

In vivo, trans-synaptic tract-tracing utilizing manganese-enhanced magnetic resonance imaging (MEMRI)

It is well established that manganese ion (Mn2+) can access neurons through voltage-gated calcium (Ca2+) channels. Based upon this fundamental principle, Mn2+ has long been used in biomedical research as an indicator of Ca2+ influx in conjunction with fluorescent microscopy. Additionally, after entry into neurons, Mn2+ is transported down axons via microtubule based fast axonal transport. Furthermore, Mn2+ is paramagnetic, resulting in a shortening of the spin-lattice relaxation time-constant, T1, which yields positive contrast enhancement in T1-weighted MRI images, specific to tissues where the ion has accumulated. Manganese-enhanced MRI (MEMRI) utilizes a combination of these properties of Mn2+ to trace neuronal pathways in an MRI-detectable manner. The focus of this review will detail some of the current MEMRI tract-tracing methodologies in mice and non-human primates as well as biological applications of MEMRI tract-tracing.     

In vivo MRI of olfactory ensheathing cell grafts and regenerating axons in transplant mediated repair of the adult rat optic nerve

The purpose of the present study was to use magnetic resonance imaging (MRI) as a tool for monitoring transplant-mediated repair of the adult rat visual pathway. We labelled rat olfactory ensheathing cells (OECs) using micron-sized particles of iron oxide (MPIO) and transplanted them by: i) intravitreal injection (ivit) and ii) intra-optic nerve (ON) injection (iON) in adult rats with ON crush (ONC) injury. We applied T(2)-weighted MRI and manganese-enhanced MRI (MEMRI) to visualise transplanted cells and ON axons at specific times after injury and cell engraftment. Our findings demonstrate that ivit MPIO-labelled OECs are unequivocally detected by T(2)-weighted MRI in vivo and that the T(1)-weighted 3D FLASH sequence applied for MEMRI facilitates simultaneous visualisation of Mn(2+-) enhanced regenerating retinal ganglion cell (RGC) axons and MPIO-labelled OEC grafts. Furthermore, analysis of MRI data and ultrastructural findings supports the hypothesis that iON OEC transplants mediate regeneration and remyelination of RGC axons post injury.