Stargazer (stg) mutant mice fail to express stargazin [transmembrane AMPA receptor regulatory protein ␥2 (TARP␥2)] and consequently experience absence seizure-like thalamocortical spike-wave discharges that pervade the hippocampal formation via the dentate gyrus (DG). As in other seizure models, the dentate granule cells of stg develop elaborate reentrant axon collaterals and transiently overexpress brain-derived neurotrophic factor. We investigated whether GABAergic parameters were affected by the stg mutation in this brain region. GABA A receptor (GABAR) ␣4 and ␤3 subunits were consistently upregulated, GABAR ␦ expression appeared to be variably reduced, whereas GABAR ␣1, ␤2, and ␥2 subunits and the GABAR synaptic anchoring protein gephyrin were essentially unaffected. We established that the ␣4␤␥2 subunit-containing, flunitrazepam-insensitive subtype of GABARs, not normally a significant GABAR in DG neurons, was strongly upregulated in stg DG, apparently arising at the expense of extrasynaptic ␣4␤␦-containing receptors. This change was associated with a reduction in neurosteroid-sensitive GABAR-mediated tonic current. This switch in GABAR subtypes was not reciprocated in the tottering mouse model of absence epilepsy implicating a unique, intrinsic adaptation of GABAergic networks in stg.Contrary to previous reports that suggested that TARP␥2 is expressed in the dentate, we find that TARP␥2 was neither detected in stg nor control DG. We report that TARP␥8 is the principal TARP isoform found in the DG and that its expression is compromised by the stargazer mutation. These effects on GABAergic parameters and TARP␥8 expression are likely to arise as a consequence of failed expression of TARP␥2 elsewhere in the brain, resulting in hyperexcitable inputs to the dentate.

Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ɛ-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell–pore interactions in situ. Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types (fibroblasts and cortical astrocytes) and how they interact with a range of pores (100–350 μm in diameter) for up to 50 days indicated that the cells either ‘covered’ or ‘bridged’ the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent “joint efforts”. Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.

Potential treatment strategies for the repair of spinal cord injury (SCI) currently favor a combinatorial approach incorporating several factors, including exogenous cell transplantation and biocompatible scaffolds. The use of scaffolds for bridging the gap at the injury site is very appealing although there has been little investigation into the central nervous system neural cell interaction and survival on such scaffolds before implantation. Previously, we demonstrated that aligned microgrooves 12.5-25 mm wide on e-polycaprolactone (PCL) promoted aligned neurite orientation and supported myelination. In this study, we identify the appropriate substrate and its topographical features required for the design of a three-dimensional scaffold intended for transplantation in SCI. Using an established myelinating culture system of dissociated spinal cord cells, recapitulating many of the features of the intact spinal cord, we demonstrate that astrocytes plated on the topography secrete soluble factors(s) that delay oligodendrocyte differentiation, but do not prevent myelination. However, as myelination does occur after a further 10-12 days in culture, this does not prevent the use of PCL as a scaffold material as part of a combined strategy for the repair of SCI.

. (2012) AbstractIn tissue engineering, the chemical and topographical cues within three-dimensional (3D) scaffolds are normally tested using static cell cultures but applied directly to tissue cultures in perfusion bioreactors. As human cells are very sensitive to the changes of culture environment, it is essential to evaluate the performance of any chemical, and topographical cues in a perfused environment before they are applied to tissue engineering. Thus the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3Dscaffolds. For this we developed a scale down bioreactor system, which allows to evaluate the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (1) quick and firm cell adhesion in 3D scaffold was critical for cell survival in perfusion culture compared with static culture, thus cell seeding procedures for static cultures might not be applicable. Therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures, (2) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion, (3) micro-grooves still maintained its influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on groovedsubstrates. This research demonstrated that the mini-bioreactor system is crucial for the development 3 of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture.