In this investigation, we report identification and characterization of a 95 kDa postsynaptic density protein (PSD-95)/discs-large/ZO-1 (PDZ) domain-containing protein termed tamalin, also recently named GRP1-associated scaffold protein (GRASP), that interacts with group 1 metabotropic glutamate receptors (mGluRs). The yeast two-hybrid system and in vitro pull-down assays indicated that the PDZ domain-containing, amino-terminal half of tamalin directly binds to the class I PDZ-binding motif of group 1 mGluRs. The C-terminal half of tamalin also bound to cytohesins, the members of guanine nucleotide exchange factors (GEFs) specific for the ADP-ribosylation factor (ARF) family of small GTP-binding proteins. Tamalin mRNA is expressed predominantly in the telencephalic region and highly overlaps with the expression of group 1 mGluR mRNAs. Both tamalin and cytohesin-2 were enriched and codistributed with mGluR1a in postsynaptic membrane fractions. Importantly, recombinant and native mGluR1a/tamalin/cytohesin-2 complexes were coimmunoprecipitated from transfected COS-7 cells and rat brain tissue, respectively. Transfection of tamalin and mutant tamalin lacking a cytohesin-binding domain caused an increase and decrease in cell-surface expression of mGluR1a in COS-7 cells, respectively. Furthermore, adenovirus-mediated expression of tamalin and dominant-negative tamalin facilitated and reduced the neuritic distribution of endogenous mGluR5 in cultured hippocampal neurons, respectively. The results indicate that tamalin plays a key role in the association of group 1 mGluRs with the ARF-specific GEF proteins and contributes to intracellular trafficking and the macromolecular organization of group 1 mGluRs at synapses.

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. A specialized medium and several growth factors were tested to study the in vitro behavior of bovine type A spermatogonia, a cell population that includes the SSCs and can be specifically stained for the lectin Dolichos biflorus agglutinin. During short-term culture (2 weeks), colonies appeared, the morphology of which varied with the specific growth factor(s) added. Whenever the stem cell medium was used, round structures reminiscent of sectioned seminiferous tubules appeared in the core of the colonies. Remarkably, these round structures always contained type A spermatogonia. When leukemia inhibitory factor (LIF), epidermal growth factor (EGF), or fibroblast growth factor 2 (FGF2) were added, specific effects on the numbers and arrangement of somatic cells were observed. However, the number of type A spermatogonia was significantly higher in cultures to which glial cell line-derived neurotrophic factor (GDNF) was added and highest when GDNF, LIF, EGF, and FGF2 were all present. The latter suggests that a proper stimulation of the somatic cells is necessary for optimal stimulation of the germ cells in culture. Somatic cells present in the colonies included Sertoli cells, peritubular myoid cells, and a few Leydig cells. A transplantation experiment, using nude mice, showed the presence of SSCs among the cultured cells and in addition strongly suggested a more than 10 000-fold increase in the number of SSCs after 30 days of culture. These results demonstrate that bovine SSC self-renew in our specialized bovine culture system and that this system can be used for the propagation of these cells.

Layer 1 in the developing cerebral cortex is populated by two basic neuronal cell types, Cajal-Retzius (CR) cells and non-CR cells. We generated transgenic mice in which green fluorescent protein (GFP) was driven by the promoter of metabotropic glutamate receptor subtype 2 and expressed specifically in CR cells during cortical development. On the basis of the precise identification of CR cells with GFP fluorescence, we pursued developmental changes and synaptic mechanisms of both CR and non-CR cells during the postnatal period. Immunostaining in combination with GFP fluorescence imaging showed that GFP and reelin, a protein involved in corticogenesis, completely overlap in CR cells at postnatal day 0. At the subsequent postnatal stage, reelin-positive neurons are segregated and categorized into GFP-positive/GABA-negative CR cells and GFP-negative/GABA-positive non-CR cells. Individual and simultaneous whole-cell recordings of CR and non-CR cells in developing cerebral slices revealed that spontaneous and electrically evoked postsynaptic currents (sPSCs and ePSCs) measured in CR and non-CR cells are differentially mediated by GABA(A) receptors versus GABA(A), AMPA, and NMDA receptors, respectively. Furthermore, CR and non-CR cells show synchronized repetitive barrages of sPSCs that reflect a network-driven activity in the developing cerebral cortex. These findings imply that the layer 1 neurons dynamically change and play a distinct and integral role in the postnatal developing neocortex.