Magnetic resonance imaging of cortical connectivity in vivo.

Magnetic resonance imaging of neuronal connectivity in vivo opens up the possibility of performing longitudinal investigations on neuronal networks. This is one main reason for the attention that paramagnetic ion manganese (Mn2+) has attracted as a potential anterograde neuronal tracer for MRI experiments. However, the correct and possibly repeated use of this tracer--or of any tracer for that matter, including heavy metals--requires the development of an administration strategy that minimizes its toxic effects. Here we first investigated the conditions that maximize the tracing efficiency of Mn2+ and preserve viability and tissue architectonics in combined MRI and histology experiments in rats. We demonstrate that most common protocols for neuronal tract tracing using Mn2+ result in large neuronal and glial lesions. The toxicity of manganese is distinct during intracortical injections and blocks the transfer of the tracer. After optimizing the technique, we could show that extensive cortical connectivity maps can be generated, with no sign of neuronal damage. Importantly, preservation of tissue viability improves the efficiency of Mn2+ in tracing neuronal connections. We have successfully used this technique to trace corticofugal somatosensory and motor pathways in individual animals and describe a connectivity index (CnI) based on Mn2+ transport that quantitatively reveals cortical heterogeneities in interhemispheric communication. Finally, we have significantly improved the resolution of the technique by continuously infusing very low concentrations of Mn2+ into the target area using osmotic pumps coupled to chronically implanted brain cannulae. The specific, nontoxic and quantitative nature of the neuronal tracings described here indicates the value of this tracer for chronic studies of development and plasticity as well as for studies of brain pathology.

Human estrogen receptor ligand activity inversion mutants: receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens.

The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.