Termination and initial branch formation of SNAP-25-deficient thalamocortical fibres in heterochronic organotypic co-cultures.

We are interested in the role of neural activity mediated through regulated vesicular release in the stopping and early branching of the thalamic projections in the cortex. Axon outgrowth, arrival at the cortical subplate, side-branch formation during the waiting period and cortical plate innervation of embryonic thalamocortical projections occurs without major abnormalities in the absence of regulated release in Snap25 (-/-) null mutant mice [Washbourne et al. (2002) Nat. Neurosci. 5:19-26; Molnár et al. (2002) J. Neurosci. 22:10313-10323]. The fact that Snap25 (-/-) null mutant mice die at birth limited our previous experiments to the prenatal period. We therefore investigated the behaviour of thalamic projections in co-culture paradigms by using heterochronic thalamic [embryonic day (E)16-E18] and cortical [postnatal day (P)0-P3] explants, in which the stopping and branching behaviour has been previously documented. Our current co-culture experiments established that thalamic projections from E16-E18 Snap25(+/+) or Snap25 (-/-) explants behaved in an identical fashion in P0-P3 Snap25 (+/+) cortical explants after 7 days in vitro. Thalamic projections from Snap25 (-/-) explants developed similar patterns of fibre ingrowth to the cortex, and stopped and formed branches at a similar depth in the Snap25(+/+) cortical slice as in control cultures. These results imply that thalamic projections can reach their ultimate target cells in layer 4, stop, and start to develop branches in the absence of regulated vesicular transmitter release from their own terminals.

The development of cortical connections.

The cortex receives its major sensory input from the thalamus via thalamocortical axons, and cortical neurons are interconnected in complex networks by corticocortical and callosal axons. Our understanding of the mechanisms generating the circuitry that confers functional properties on cortical neurons and networks, although poor, has been advanced significantly by recent research on the molecular mechanisms of thalamocortical axonal guidance and ordering. Here we review recent advances in knowledge of how thalamocortical axons are guided and how they maintain order during that process. Several studies have shown the importance in this process of guidance molecules including Eph receptors and ephrins, members of the Wnt signalling pathway and members of a novel planar cell polarity pathway. Signalling molecules and transcription factors expressed with graded concentrations across the cortex are important in establishing cortical maps of the topography of sensory surfaces. Neural activity, both spontaneous and evoked, plays a role in refining thalamocortical connections but recent work has indicated that neural activity is less important than was previously thought for the development of some early maps. A strategy used widely in the development of corticocortical and callosal connections is the early overproduction of projections followed by selection after contact with the target structure. Here we discuss recent work in primates indicating that elimination of juvenile projections is not a major mechanism in the development of pathways feeding information forward to higher levels of cortical processing, although its use is common to developing feedback pathways.

Electron and atomic force microscopy of the trimeric ammonium transporter AmtB.

Escherichia coli AmtB is an archetypal member of the ammonium transporter (Amt) family, a family of proteins that are conserved in all domains of life. Reconstitution of AmtB in the presence of lipids produced large, ordered two-dimensional crystals. From these, a 12 A resolution projection map was determined by cryoelectron microscopy, and high-resolution topographs were acquired using atomic force microscopy. Both techniques showed the trimeric structure of AmtB in which each monomer seems to have a pseudo-two-fold symmetry. This arrangement is likely to represent the in vivo structure. This work provides the first views of the structure of any member of the Amt family.